Specific murine and humanized monoclonal antibodies detecting pathology associated secondary structure changes in proteins and peptides

ABSTRACT

The present invention relates to antibodies and binding fragments thereof that bind the β-sheet secondary structure of a pathological monomeric or oligomeric non-fibrillar proteins without binding to the non-toxic, non-pathological forms of these proteins or peptides. These antibodies and binding fragments thereof are suitable for the diagnosis, prevention, and treatment of protein conformational disorders including all amyloid diseases.

This application claims the priority benefit of U.S. Provisional PatentApplication Ser. No. 62/365,465, filed on Jul. 22, 2016.

This invention was made with government support under grant numberNS073502 awarded by the National Institutes of Health. The governmenthas certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to antibodies and binding fragmentsthereof that bind β-sheet secondary structures on pathological proteins,such as the β-sheet secondary structures found in toxic oligomeric formsof amyloidogenic proteins or in infectious, toxic or necrotic proteincomplexes. These antibodies and binding fragments thereof are suitablefor the diagnosis, prevention, and treatment of protein conformationaldisorders including all amyloid disease and some foreign infectivecomplexes.

BACKGROUND OF THE INVENTION

Amyloidosis broadly encompasses a variety of diseases that arecharacterized by the extracellular or intracellular deposition ofamyloid proteins in tissues and/or organs. Amyloidosis starts with theconformational change of an otherwise soluble physiologicalpeptide/protein into a pre-pathological or pathological conformer thathas acquired a higher percentage of β-sheet structure, which is evidentby the secondary and/or the tertiary structure of the peptide/protein.This conformational change leads to the generation of a toxic oligomericform and ultimately to a fibrillary structure that becomes insoluble andprecipitates in the milieu where the change was initiated. Thus, aninitial conformational disorder might end up being an amyloid diseaseand/or a toxic conformational disease of pre-fibrillar amyloidoticmaterial (Wisniewski and Gail, “Immunotherapy for Alzheimer's Disease,”Biochem Pharmacol 88:499-507 (2014)). Amyloids are insolubleprotein/peptide aggregates in fibrillar form, and their deposition mayoccur in localized sites or systemically. The fibrillar composition ofthese deposits is an identifying characteristic for the various forms ofamyloid disease. In some cases the amyloid protein/peptide accumulatesintracellularly, resulting in cell dysfunction and ultimately celldeath. Examples of intracellular amyloid proteins include, among others,α-synuclein, forming Lewy bodies in Parkinson's disease, and huntingtin,forming neuronal inclusions in Huntington disease. The pathogenesis ofAlzheimer's disease (AD), the most common of the conformational amyloidrelated neurodegenerative disorders, is linked to the forming of twodifferent pathological conformers. The first of these is characterizedby the cleavage of the amyloid precursor protein (APP) generatingamyloid-β (Aβ) peptides of about 30 to 55 amino acids, which undergo ashape change into a pathological conformer having high 13 sheet content.Intracerebral and cerebrovascular deposits composed primarily of fibrilsof the pathological Aβ peptide are characteristic of both familial andsporadic forms of AD. In addition to Aβ, conformationally abnormalhyper-phosphorylated tau protein forms toxic oligomeric structures andintraneuronal deposited neurofibrillary tangles in AD as well as infronto-temporal dementias. Similar to AD, prion-associated diseases,such as Creutzfeldt-Jacob disease, have also been characterized asamyloid diseases. The pathogenesis of prion disease is linked to aconformational change of the cellular prion protein (PrP^(C)) into thedisease associated PrP^(Sc) (Sc for scrapie). Currently there is noeffective therapy for any of these disorders.

An active area of translational research and current clinical trials foramyloid disease has focused on immunotherapy, using both passive andactive immunization against amyloid proteins, particularly Aβ in AD(Wisniewski and Goñi, “Immunotherapeutic Approaches for Alzheimer'sDisease,” Neuron 85:1162-1170 (2015)). Although conventional anti-Aβspecific immunotherapy held great promise as a means of reducing amyloiddeposition, it, unfortunately, has been accompanied by major obstacles.Specific problems associated with immunotherapy that were identified ina clinical trial for AD include the potential of toxicity fromencephalitis (related to excessive cell mediated immunity), theimmunological targeting of both the normal and abnormal Aβ peptide, thefailure to address tau related pathology, and the apparent poorefficacy. Moreover, although autopsy data from this early immunotherapyvaccine trial suggested that many patients had a significant reductionin amyloid burden, these patients exhibited only minor cognitivebenefits (Wisniewski et al., “Amyloid-β Immunization for Alzheimer'sDisease,” Lancet Neurol 7:805-811 (2008) and Holmes et al., “Long TermEffects of Aβ42 Immunization in Alzheimer's Disease: Immune Response,Plaque Removal and Clinical Function,” Lancet 372:216-223 (2008)).Therefore, an immunotherapeutic approach directed specifically to theβ-sheet conformation that can effectively reduce the toxic oligomericforms and amyloid burden of both Aβ and tau and overcome theaforementioned problems is warranted.

The present invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

A first aspect of the present disclosure is directed to an antibody orbinding fragment thereof that binds β-sheet secondary structure of apathological monomeric or oligomeric non-fibrillar protein. The antibodyor binding fragment thereof comprises a heavy chain variable region thatcomprises a complementarity-determining region 1 (H-CDR1) having anamino acid sequence of any one of SEQ ID NOs: 23-26, and 50, or amodified amino acid sequence of any one of SEQ ID NOs: 23-26, and 50,said modified sequence containing 1, 2, or 3 amino acid residuemodifications as compared to any one of SEQ ID NOs: 23-26, or 50. Theheavy chain variable region also comprises a complementarity-determiningregion 2 (H-CDR2) having an amino acid sequence of any one of SEQ IDNOs: 27-30, and 51, or a modified amino acid sequence of any one of SEQID NOs: 27-30, and 51, said modified sequences containing 1, 2, 3, or 4amino acid residue modifications as compared to any one of SEQ ID NOs:27-30, or 51. The heavy chain variable region further comprises acomplementarity-determining region 3 (H-CDR3) having an amino acidsequence of any one of SEQ ID NOs: 31-34, and 52, or a modified aminoacid sequence of any one of SEQ ID NO: 31-34, and 52, said modifiedsequence containing 1, 2, or 3 amino acid residue modifications ascompared to any one of SEQ ID NOs: 31-34, and 52.

The antibody or binding fragment thereof as described herein may furthercomprise a light chain variable region. The light chain variable regioncomprises a complementarity-determining region 1 (L-CDR1) having anamino acid sequence of any one of SEQ ID NOs: 35-39, and 53, or amodified amino acid sequence of any one of SEQ ID NO: 35-39, and 53,said modified sequence containing 1, 2, 3, or 4 amino acid residuemodifications as compared to any one of SEQ ID NO: 35-39, or 53. Thelight chain variable region also comprises a complementarity-determiningregion 2 (L-CDR2) having an amino acid sequence of any one of SEQ IDNOs: 40-44, and 54, or a modified amino acid sequence of any one of SEQID NO: 40-44, and 54, said modified sequence containing 1 or 2 aminoacid residue modifications as compared to SEQ ID NO: 40-44, or 54. Thelight chain variable region also comprises a complementarity-determiningregion 3 (L-CDR3) having an amino acid sequence of any one of SEQ IDNOs: 45-49, and 55, or a modified amino acid sequence of any one of SEQID NO: 45-49, and 55, said modified sequence containing 1 or 2 aminoacid residue modifications as compared to SEQ ID NO: 45-49, or 55.

Another aspect of the present disclosure is directed to a method ofinhibiting onset of one or more symptoms of a condition mediated by anamyloidogenic protein or peptide in a subject. This method involvesadministering to the subject a pharmaceutical composition comprising anantibody or binding fragment thereof as described herein, where thecomposition is administered in an amount effective to inhibit onset ofone or more symptoms of the condition mediated by the amyloidogenicprotein or peptide in the subject.

Another aspect of the present disclosure is directed to a method oftreating a condition mediated by an amyloidogenic protein or peptide ina subject. This method involves administering to the subject apharmaceutical composition comprising an antibody or binding fragmentthereof as described herein, where the composition is administered in anamount effective to treat the or ameliorate the condition, or one ormore symptoms thereof, mediated by the amyloidogenic protein or peptidein the subject.

Another aspect of the present disclosure is directed to a method oftreating a subject having or at risk of having a condition mediated by apathological protein having a (3-sheet secondary structure. This methodinvolves administering to the subject a pharmaceutical compositioncomprising an antibody or binding fragment thereof as described herein,where the composition is administered in an amount effective to treat orameliorate the condition, or one or more symptoms thereof, mediated bythe pathological protein having the β-sheet secondary structure.

Another aspect of the present disclosure is directed to a method ofdiagnosing an amyloid disease in a subject. This method involvesdetecting, in the subject, the presence of an amyloidogenic protein orpeptide using a diagnostic reagent, wherein the diagnostic reagentcomprises the antibody or binding fragment described herein anddiagnosing the amyloid disease in the subject based on said detecting.

Another aspect of the present disclosure is directed to a method ofidentifying a subject's risk for developing a condition mediated by anamyloidogenic protein or peptide. This method involves detecting, in thesubject, the presence of an amyloidogenic protein or peptide using adiagnostic reagent comprising the antibody or binding fragment thereofdescribed herein, and identifying the subject's risk of developing thecondition mediated by the amyloidogenic protein or peptide based on theresults of the detecting step.

Another aspect of the present disclosure is directed to a diagnostic kitthat comprises the antibody or binding fragment thereof as describedherein and a detectable label.

Described herein is the development of a methodology to produceconformational anti-secondary structure β-sheet monoclonal antibodies.The β-sheet secondary structure of proteins can be derived from manydifferent primary sequences, but generally is dominant in the productionof any pathologic misfolded proteins or peptides. A small 13 amino acidspeptide of the carboxyl terminus of the very rare British amyloidosis(ABri), which is derived from an intronic DNA sequence expressed by amissense mutation and has no sequence homology to any other mammalianprotein was used as the immunogen (Wisniewski and Goñi,“Immunotherapeutic Approaches for Alzheimer's Disease” Neuron85:1162-1176 (2015), Vidal et al., “A Stop-Codon Mutation in the BRIGene Associated with Familial British Dementia” Nature 399:776-781(1999), Rostagno et al., “Chromosome 13 Dementias” Cell Mol. Life Sci62:1814-1825 (2005), and Goñi et al., “Immunomodulation TargetingAbnormal Protein Conformation Reduces Pathology in a Mouse Model ofAlzheimer's Disease” PLoS ONE 5:e13391 (2010), each of which is herebyincorporated by reference in its entirety). The peptide was polymerizedby an extensive glutaraldehyde reaction to form immunogenic, covalentlybound 10-100 kDa soluble and stable oligomers with high β-sheetsecondary structure content (p13Bri) (Goñi et al., “ImmunomodulationTargeting Abnormal Protein Conformation Reduces Pathology in a MouseModel of Alzheimer's Disease” PLoS ONE 5:e13391 (2010), and Goñi et al.,“Immunomodulation Targeting both Aβ and tau Pathological ConformersAmeliorates Alzheimer's Disease Pathology in TgSwDI and 3×Tg MouseModels” Journal of Neuroinflammation 10:150 (2013), both of which arehereby incorporated by reference in their entirety). Inoculation in micewith a suitable adjuvant p13Bri produced an array of antibodies to thenon-self motif and the β-sheet secondary structure. Hybridomas wereproduced and monoclonals were selected by the novel approach ofspecifically using as selector compounds, oligomeric conformers fromdifferent neurodegenerative disease (NDD) with the only commonalitybeing the shared β-sheet secondary structure. These new monoclonals toβ-sheet conformation in oligomers more effectively detect, monitor andtreat NDD in humans and other susceptible animals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C depict the production of anti β-sheet secondary structureconformational monoclonal antibodies with specificity to oligomerictoxic conformers present in neurodegenerative diseases (NDD). FIG. 1Ashows color coded pathways to oligomeric forms and fibrillar deposits ofself-antigenic protein/peptides associated with pathology on most commonNDD: Aβ (red) and tau (brown) for Alzheimer's Disease; α-synuclein(orange) for Lewy Body diseases, and PrP (grey) for prionoses. Blackshape represents common to all β-sheet secondary structure acquiredduring pathological conformational change. Electron microscopy (EM) ofoligomers and fibrils on the left and immunoblots of oligomeric formsdetected by specific antibodies on the right, all color coded (also inFIGS. 3A-3C, 6A-6D, and 7A-7D). FIG. 1B shows one letter code of the 13amino acids sequence of the non-self ABri peptide (purple boxed). Bottompathway shows the normal conversion of ABri peptides to oligomers andfibrils (purple). Top pathway shows the controlled polymerized reactionwith glutaraldehyde (see Example 1) leading to p13Bri the immunogenic,non-self, soluble and stable β-sheet oligomers of 10-100 kDa molecularweight (from purple to green frame). Black shapes represent common toall β-sheet structure. On the left, EM of the oligomeric p13Bri (greenframe) and the oligomer/fibrils of the aged ABri peptide (purple frame).On the right Immunoblot with rabbit polyclonal anti-Bri and circulardichroism analysis of both forms (color coded and also in FIGS. 4A-4C).FIG. 1C shows the p13Bri (green boxed) inoculated into mice to producehybridomas (Methods and table 1); horizontal blue arrows show theselection process of monoclonals by the oligomeric β-sheet conformers of└A┘ antigens (thick black frame and arrow); framed in blue the selectedanti-conformational monoclonal antibodies that recognize β-sheetsecondary structure common to [A] and [B] pathways. The thick bluearrows from the framed blue antibodies signal possible interactive siteswith pathological conformers on NDD (also in FIGS. 6A-6D, 7A-7D, and10A-10B).

FIGS. 2A-2D depict the characterization of the ABri and Polymerizedimmunogenic 13-mer Bri (p13Bri) Peptides. FIG. 2A shows electronmicroscopy (EM) of the sequential fibrillization of the ABri peptide; 1)24 hours incubation at room temperature (RT); 2) one week incubation atRT with associated fibrils; 3) and 4) two weeks incubation at RT, bigclusters of precipitated long fibrils. Scale bars represent 200 μm. FIG.2B shows EM images of 13-mer Bri peptide after controlled polymerizationwith glutaraldehyde (p13Bri) showing different oligomeric states; 5) twohours after preparation of the sample; 6) one week after incubation atRT and, 7) and 8) after two weeks of incubation at RT. Scale barsrepresent 200 μm. FIG. 2C shows nitrocellulose blot from 15% SDS-PAGE ofthe ABri (lane 1) and p13Bri (lane 2) peptides. Left panel, fast greenprotein reversible stain; right panel immunoblot with anti-Bri antibody.Bracket marks oligomeric state between 10-100 kDa. FIG. 2D showscircular dichroism of the p13Bri peptide all β structure (p13Bri) andABri monomer (ABrim).

FIGS. 3A-3C depicts immunochemistry of plasma from p13Bri immunized 3×TgAD mice and CD-1 M4 mouse on old 3×Tg AD mouse model brains, human ADand control brains; and immunoreactivity of M4 plasma on Aβ40/42 ELISA.FIG. 3A shows similarities of plasma reactivity from 3×Tg micesuccessfully immunized with p13Bri (top) 22 and CD-1 M4 mouse (bottom)on cerebral cortex and Hippocampus of old 3×Tg AD mice with amyloid andtau pathology. Right panels show higher magnification of the boxedareas. All scale bars represent 50 μm. FIG. 3B shows the same plasmacomparison as in FIG. 3A but in the cortex of human AD brains. Leftpanels show negative reactivity on human control brains. Right panelsshow magnification of the boxed areas. Arrowheads show glial-like cells;arrows show cytoplasmic punctuated staining in neurons. Scale barsrepresent 50 μm. FIG. 3C shows EM images of Aβ1-40 and Aβ1-42 peptideson 50 mM bicarbonate pH 9.6 used to coat ELISA plates. Arrows showoligomeric forms decorating amyloid fibrils in both cases. Scale barsrepresent 200 μm. Right panel shows ELISA differential IgM reactivity toAβ1-40 and Aβ1-42 from plasma of the M4 CD-1 mouse at different bleedingtimes as per Table 10.

FIGS. 4A-4C depict plasma levels of Anti-Aβ 1-40 and Anti-Aβ 1-42 fromp13Bri inoculated CD-1 mice. FIG. 4A shows ELISA data showing plasmareactivity to Aβ40 and Aβ42 from different bleedings of all CD-1 mice asper Table 11, using peroxidase-labelled goat anti-mouse IgM (μ chain).Samples were run on triplicate. FIG. 4B shows ELISA data showing plasmareactivity to Aβ40 and Aβ42 from different bleedings of all CD-1 miceusing peroxidase-labelled goat anti-mouse IgG (H+L). Samples were run ontriplicate. FIG. 4C shows plate coating control showing similarreactivity detected for Aβ40 and Aβ42 with commercial antibodies4G8/6E10 and secondary anti-mouse IgG (H+L).

FIGS. 5A-5B depict co-localization on human AD brains of IgM and IgGantibodies present in the plasma of the p13Bri inoculated CD-1 M4 mouse.FIG. 5A shows representative images showing the co-localization on thecortex of human AD brains of IgM and IgG antibodies present in theplasma of CD-1 M4 mouse inoculated with p13Bri. A combined T6+Tf poolwas used as per Table 10. FIG. 5B shows higher magnification of theboxed area in FIG. 5A.

FIGS. 6A-6D depict electron microscopy of paired helical filaments (PHF)and PKA treated PHF, and comparative detection of specific NDDconformers. FIG. 6A shows EM of purified PHF from a human AD brain andprotein kinase A treated PHF. Arrowheads show fibrils and arrows showoligomers in different aggregation clusters. Left two panels showoverall differences of fibrils and oligomers associated to fibrils or inindependent clusters. All scale bars are 50 μm. FIG. 6B showsimmunoblots of recombinant deer PrP (dPrP); Aβ1-42 freshly dissolved,fibrilized or polymerized (Aβ42, Aβ4f and Aβ42p respectively), and PHFand PKA PHF. Individual specificity of commercial antibodies PHF-1 forhyperphosphorylated tau; 4G8 and 6E10 for Aβ peptides and 7D9 and 6D11for PrP protein is compared to the cross-reactivity of hybridomas 10Eand 23B reactive to more than one conformer and oligomeric forms. FIGS.6C and 6D show co-localization of hybridoma 23B with either 4G8/6E10 orPHF-1 antibodies on human AD brain tissue. Scale bars represent 100 μm.

FIGS. 7A-7D depict electron microscopy of fibrillar and oligomeric formsof misfolded protein/peptides from NDDs and the recognition in ELISA andimmunoblots by the original hybridomas, before subcloning, 3D, 23B, 10E,11F, 10F, and 12E. FIG. 7A shows EM of Aβ1-40, Aβ1-42, PHF purified froma human AD brain, and oligomerized PrP; arrows show oligomeric formsdecorating amyloid fibrils. The right panel shows the ELISA reactivityof original positive or irrelevant clones to four neuroconformers. FIG.7B (top) shows EM of Aβ1-40 and Aβ1-42 polymerized with glutaraldehydeand Aβ1-42 fibrillized, and the corresponding immunoblots. FIG. 7B(bottom) shows molecular weight marker (MW) followed by Aβ1-40polymerized (Aβ40p), Aβ1-42 freshly dissolved (Aβ42), Aβ1-42 fibrillized(Aβ42f) and Aβ1-42 polymerized (Aβ42p). The left panel shows reversibleFast Green (FG) protein stain to assess comparable protein load. Thenext panel shows the reactivity with commercial IgG monoclonals 4G8 and6E10 specific for Aβ peptides sequence. The three right panels showpositive original clones before subcloning with differential reactivityto oligomeric forms of Aβ. FIG. 7C (top) shows EM of α-synucleinmonomer, fibrillized on PBS or oligomerized with glutaraldehyde, andPHF. Bottom panels corresponding immunoblots lanes: α-synuclein monomer(α-syn m), α-synuclein fibrillized (α-syn f), α-synuclein oligomerized(α-syn p) and PHF. Left panel FG, next panel commercial anti-α-synucleinantibody; third from left commercial PHF-1 and to the right fiveoriginal clones. FIG. 7D (top) shows EM of different states ofaggregation of aged recPrP molecules. Bottom, immunoblots lanes: human(HuPrP), sheep (ShPrP) and deer PrP (dPrP). All PrPs were incubated forat least two days to maximize aggregation. Left panel FG, second panelcommercial IgG monoclonals 7D9 and 6D11 that recognize middle parts ofPrP. Next five panels show the reactivity of 5 original clones, beforesubcloning, with differential oligomer size detection previously unseen.

FIGS. 8A-8B depict control of protein/peptide coat and IgM reactivity inELISA plates of Aβ1-40, Aβ1-42 and PHF. FIG. 8A shows typical ELISAplates coated with (in order from left to right on the graph) Aβ1-40,Aβ1-42, or PHF as per FIGS. 6A-6D, showing the difference between aclone with potential conformational monoclonal antibody and anirrelevant clone to mark the background IgM reactivity on a cellsupernatant that had comparable number of cells. FIG. 8B shows controlof the coat in each ELISA plate with commercial mouse IgG anti-Aβ 4G8and 6E10 and commercial IgG anti-PHF PHF-1 (bars with lines). Backgroundanti-IgM reactivity is similar to the irrelevant clone (hatched bars).

FIGS. 9A-9D depict purification and characterization of potential antiβ-sheet secondary structure conformational monoclonal antibodiesobtained from the fusion of p13Bri hyper-immunized M4 CD-1 mouse spleencells with SP2/mIL-6 fusion partner. FIG. 9A shows fast green of cellsupernatants of original hybridomas 3D, 10E, 10F, 11F, 12E and 23Bobtained from the fusion of spleen cells of p13Bri immunized M4 mouseand SP2/0-IL6 fusion partner. Large amount of bovine serum albumin (BSA)from the growth media supplementation is shown. FIGS. 9B-9D show westernblot of the 40% saturated Ammonium Sulfate (SAS) purified antibodiesfrom original clones 3D, 10E, 10F, 11F, 12E and 23B. FIG. 9B shows fastgreen stain with residual BSA. FIG. 9C shows anti-mouse IgM preactivity. FIG. 9D shows anti-mouse Kappa reactivity. Left part of theblots show untreated samples and right part 0.1M Dithiothreitol (DTT)disulfide bridges reduced samples. IgMk p: pentameric IgM; IgMk m:monomeric IgM; Hμ r: mu Heavy chain reduced; Kf: free kappa Light chainsand Kr: reduced kappa light chains.

FIGS. 10A-10B depict immunohistochemistry of plasma from p13Briimmunized M4 mouse and five partially purified potential anti β-sheetsecondary structure conformational monoclonal antibodies on human AD andcontrol brains. FIG. 10A shows immunolabeling of human AD brain cortex.FIG. 10B shows human control brain cortex without pathology. Left panel,reactivity of the plasma from p13Bri immunized M4 mouse, arrows showcytoplasmic staining that extends to processes. Next five panelsammonium sulfate semi-purified anti β-sheet secondary structureconformational monoclonals. 23B labels cytoplasm, processes andextracellular material; 3D labels the whole neuronal body; 12E showspreference for glial cells; 10E and 10F show similar lighter stainingpattern of neuronal cytoplasm, processes and nucleus. Scale barsrepresent 50 μm.

FIGS. 11A-11B depict purification of the conformational monoclonalantibody WG-3D7.

FIGS. 12A-12E show electron microscopy images of aggregated/oligomericAmyloid β and PHF, and reactivity of the conformational monoclonalantibody WG-3D7 against these same pathogenic peptides.

FIGS. 13A-13B demonstrate binding of the conformational monoclonalantibody WG-3D7 to oligomeric Aβ42 by surface plasmon resonance.

FIGS. 14A-14B depict immunohistochemistry on AD, age-matched, and youngcontrol human brains sections using conformational monoclonal antibodyWG-3D7.

FIGS. 15A-15B depict immunohistochemistry of conformational monoclonalantibody WG-3D7 on human AD brains sections.

FIGS. 16A-16B demonstrate co-localization of conformational monoclonalantibody WG-3D7 and pathological Tau species in human AD brain sections.

FIGS. 17A-17B depict purification of the conformational monoclonalantibody FT-11F2 with SAS and quantification of the antibody bindinglevels to pathological conformers.

FIGS. 18A-18B demonstrate the reactivity of the conformationalmonoclonal antibody FT-11F2 against normal, fibrilized, and oligomericα-synuclein and fibrils of purified human PHF.

FIG. 19 depicts immunoreactivity of the conformational monoclonalantibody FT-11F2 against oligomeric α-synuclein forms surrounding Lewybodies in Parkinson's disease in human brains.

FIG. 20 depicts FT-12E1 reactivity against (from left to right on graph)Aβ 1-40, oligomerized Aβ 1-42, human purified PHF, and oligomerized,recombinant deer Prion protein (dPrP) (left graph).

FIGS. 21A-21B depict purification of the conformational monoclonalantibody FT-12E1.

FIGS. 22A-22C show the reactivity of the conformational monoclonalantibody FT-12E1 against purified human PHF, three different strains ofpathogenic PrP^(Res) and α-synuclein.

FIGS. 23A-23B demonstrate conformational monoclonal antibody FT-12E1reactivity in AD human brain sections.

FIGS. 24A-24C demonstrate conformational monoclonal antibody FT-12E1immunoreactivity in AD human brain sections.

FIGS. 25A-25B show western blots of the conformational monoclonalantibodies TF-10E8 and TF-10F7 purified with a llama anti-μ column.

FIGS. 26A-26C show electron microscopy images of purified human PHF andreactivity of the conformational monoclonal antibodies TF-10E8 andTF-10F7 against PHF and three different strains of pathogenic PrP.

FIGS. 27A-27B depict immunoreactivity of the conformational monoclonalantibody TF-10E8 in AD affected human brain hippocampus tissue.

FIGS. 28A-28B depict immunoreactivity of the conformational monoclonalantibody TF-10F7 in AD affected human brain sections.

FIGS. 29A-29B depict immunoreactivity of the conformational monoclonalantibodies TF-10E8 and TF-10F7 in human brain sections of a subjecthaving Gerstmann-Straussler-Scheinker syndrome (GSS), a genetic,autosomal dominant prion disease.

FIGS. 30A-31E depict reactivity of the original hybridoma 23B selectedclone against Aβ1-40, Aβ1-42 and PHF; and the partial purification ofthe sub-clone GW-23B7 with saturated ammonium sulfate (SAS). FIG. 30Ashows electron microscopy images of Aβ40 and Aβ42 in ammoniumbicarbonate pH 9.6, used to coat ELISA plates. Arrows showrepresentative oligomeric forms around fibrils of Aβ40 and Aβ42respectively; scattered and loose in Aβ40 and compact and bundled inAβ42. Bars represent 100 μm. FIG. 30B shows ELISA assay showingcross-reactivity of the cell supernatant of hybridoma 23B clone to (fromleft to right on the graph) Aβ1-40, Aβ1-42 and PHF detected by ananti-mouse IgG+IgA+IgM (H+L) antisera. FIG. 30C shows Fast Green of theconcentrated cell supernatant, unreduced and DTT reduced Lanes 1 and 2,obtained from the sub-cloned conformational mAb GW-23B7 beforepurification and dominated by Bovine serum albumin (BSA) from fetal calfserum. FIG. 30D shows Fast Green of the 30% SAS precipitate showing theintact IgM and the Heavy and Light chains before and after reductionrespectively (Lanes 3 and 4), and a small amount of the remaining BSA.IgMk p: pentameric; IgMk m: monomeric; Heavy chain reduced (Hμ r);truncated Heavy chain reduced (Hμ t), and Kappa Light chain reduced (Kr)shown. FIG. 30E shows Immunoblot of the SAS partially purifiedconformational mAb GW-23B7. The anti-mouse IgM (p, chain specific) showsthe intact pentamer and IgM monomer before reduction (Lane 3) and afterreduction the Heavy chain intact around 76 kDa plus 10-15% of atruncated Heavy chain at 60 kDa (Lane 4). The anti-mouse kappa antibodyshows its presence in the pentameric and slightly in the monomeric IgMbefore reduction (Lane 3′) and only one band for the Kappa Light chainafter reduction (Lane 4′).

FIGS. 31A-31D depict sub-cloned and purified anti-β-sheet conformationalmonoclonal antibody (aβComAb) GW-23B7 specific reactivity to humanpaired helical filaments (PHF) and oligomeric Aβ. FIG. 31A shows ELISAdata showing the reactivity from cell supernatant of sub-cloned GW-23B7IgM and an irrelevant clone from the same fusion, to PHF and oligomersdifferential on Aβ1-40 and Aβ1-42 (see Example 6). The right panel showsthe even coating of the selected peptides on the plate and the lack ofunspecific reactivity to secondary anti-mouse IgM. FIG. 31B showswestern blot to show the pentameric integrity of the purified aβComAbGW-23B7. Lane 1 unreduced sample, lane 2 reduced with 0.1M DTT. Leftpanel: Fast Green reversible protein stain; middle panel: anti-mouse IgM(μ specific) and right panel: anti-mouse Kappa Light chains. IgMp:pentameric immunoglobulin M; Hμr: μ heavy chain reduced; Kr: Kappa lightchain reduced. FIG. 31C shows ELISA assay showing the reactivity ofpurified aβComAb GW-23B7 diluted 1:1000, to (from left to right on thegraph) Aβ1-40, Aβ1-42 and human PHF. FIG. 31D shows Surface PlasmonResonance showing the binding affinity of the purified aβComAb GW-23B7to the oligomeric species of Aβ1-42 and the lack of binding affinity tothe monomeric forms. The K_(D) (14 nM) was determined from the raw dataon the left.

FIGS. 32A-32D depict histochemical reactivity of aβComAb GW-23B7 toAlzheimer's disease (AD), age matched and young control human brains,and Gerstmann-Straussler-Scheinker (GSS) prion disease; and recognitionof pathological conformers on immunoblots. FIG. 32A shows representativeimages of the immunohistochemistry showing reactivity of the aβComAbGW-23B7 on human AD brains (top panels) compared to aged-matched andyoung human brain controls (middle and bottom panels respectively).Right panels are magnifications of the boxed areas on the left. Scalebars represent 100 μm on the left panels and 50 μm on the right panels.FIG. 32B shows fluorescent immunohistochemistry on human AD brains (twotop panels) and a GSS brain (bottom panels). Left panels: GW-23B7 (leftchannel); middle panels (middle channels) commercial antibodies4G8/6E10, PHF-1 and anti-glial fibrillary acidic protein (GFAP) top tobottom respectively; and right panels showing the co-localizationindicated by white arrows. Scale bars represent 50 μm. FIG. 32C shows EMof Aβ1-42 fibrilized or polymerized with glutaraldehyde, PHF and proteinkinase A treated PHF used on SDS-PAGE for immunoblots. FIG. 32D showsimmunoblots comparing reactivity of the aβComAb GW-23B7 with thespecific anti-Aβ peptides antibodies 4G8/6E10 to Aβ1-40, Aβ1-40polymerized, Aβ1-42 fibrilized and Aβ1-42 polymerized (Lanes 1 to 4respectively); the PHF-1 specific antibody for hyperphosphorylated tauon PHF and PHF PKa treated (Lanes 5 and 6 respectively), and the7D9/6D11 antibodies specific for PrP molecules to oligomerized PrP andCWD prions (Lanes 7 and 8 respectively).

FIGS. 33A-33C depict conformational monoclonal antibody GW-23B7immunoreactivity on human Alzheimer's disease brains. All slides arefrom human AD brains; FIG. 33A) and FIG. 33B) cortex and FIG. 33C)Hippocampus. The conformational mAb GW-23B7 was used as primary reagentdetected by HRP-labeled anti-mouse IgM (μ specific) as a secondaryantibody; color was developed using DAB. Right panels are magnificationsof the boxed areas on the left. Bars represent 50 μm. FIGS. 33A-33C showvarious potential stages of extra- and intracellular pathology areapparently detected by GW-23B7. Dark stain is seen in the cytoplasm ofmany neurons with some of them extending punctuate stain inside thenucleus (white arrows). FIG. 33A shows extensive punctuatedextra-cellular material (black arrows) coincidental within supposedplaque contours; many neurons seem to be dystrophic or degraded to apoint that they lose membrane definition and integrity (blackarrowheads). Neuronal processes are also detected (white arrowheads).Some in FIG. 33B apparently making contact through zones of defined orundefined intact structure; whereas in FIG. 33C all the length of thedetected material is confined in either well-defined cellular structuresas in the neuron on the bottom right (right panel) or in discontinuousstructures “leaking” to the extracellular milieu from the dystrophicneuron on the upper left.

FIGS. 34A-34C depicts molecular integrity and AD pathology recognitionof the purified AβComAb GW-23B7 infused on old 3×Tg mice. FIG. 43A showsimmunoblot showing protein stain, μ and kappa reactivity around the1,000 kDa position of an intact pentameric IgMk (Lane 1). FIG. 34B showsimmunoblot of PHF and PHF PKa purified from a human AD brain (Lanes Aand B) detected by PHF-1 commercial antibody specific forhyperphosphorylated tau or by the GW-23B7; right panel showsimmunohistochemical recognition by GW-23B7 of intra- and extracellularpathology associated structures on a human AD brain section. FIG. 34Cshows immunoblots comparing reactivity to Aβ1-42 and Aβ1-42 oligomerized(Lanes C and D) by the commercial anti-Aβ 4G8/6E10 antibodies or theGW-23B7; on the right panel co-localization on a human AD brain sectionof punctuated or strong precipitated material recognized by GW-23B7(black stain) within amyloid plaques of Aβ amyloid detected by 4G8/6E10specific antibodies (light grey stain).

FIGS. 35A-35C depict a protocol of infusion of aβComAb GW-23B7 orvehicle control on 3×Tg mice and the ensuing behavioral and kineticstests. FIG. 35A shows protocol of the intra-peritoneal infusion ofGW-23B7 or vehicle alone on 3×Tg AD mice, behavioral tests and sacrificeof the tested animals. FIG. 35B shows comparative kinetics of pentamericIgM distribution inside brains pooled from 18 m.o. 3×Tg animals infusedwith either GW-23B7 or vehicle alone as per bottom part of protocol onFIG. 35A. Western blots of soluble supernatants from 20% brainhomogenates were stained before and after reduction with reversible FastGreen to assess comparable protein loading (top panel) and detected byanti-mouse μH chain or anti-mouse kappa L chain on the middle and bottompanel respectively. The relative concentrations determined bydensitometry of the bands are plotted on the graph on the right. (c).Radial Arm Maze (RAM) behavioral test showing significant differences(p<0.0001 determined by two-way ANOVA) between animals infused withGW-23B7 (n=4) or vehicle alone (n=4). In the treated animal group therewas also a significant days effect (p<0.0001).

FIGS. 36A-36B depict locomotor tests on 18 m.o. 3×Tg AD mice infusedwith aβComAb GW-23B7 or with control vehicle alone. FIG. 36A showsRotarod to determine balance and coordination; no differences were seenbetween the control and the GW-23B7 infused groups. FIG. 36B showsTraverse Beam to determine general motor coordination; no differenceswere seen between the control and the GW-23B7 infused groups.

FIGS. 37A-37C depict levels of extracellular amyloid-β burden,intracellular PHF burden and soluble Aβ or ptau on brains from 19 monthsold 3×Tg mice infused with aβComAb GW-23B7 or control vehicle alone.FIG. 37A shows immunohistochemistry of representative GW-23B7 or vehiclecontrol infused 3×Tg mouse brains showing on Hippocampus and Subiculumamyloid plaques detected by specific antibodies 4G8/6E10 orintraneuronal PHF as detected by commercial antibody PHF-1. Scale barsrepresent 200 μm. FIG. 37B shows quantitation of the amyloid and tauburden for each group of infused animals characterized on FIG. 37A; *:p<0.05 and **: p<0.01. No significant differences for intracellularPHF-1 burden. FIG. 37C shows levels of soluble Aβ1-40, Aβ1-42, humanaggregated Aβ, threonine 231 phosphorylated tau and total tau onsupernatants from 20% brain homogenates of the GW-23B7 and vehicleinfused 3×Tg mice groups; *: p<0.05 and ****: p<0.0001.

FIGS. 38A-38B depict immunoblots detecting oligomers on solublesupernatants from 20% brain homogenates of 19 m.o. 3×Tg mice infusedwith purified aβComAb GW-23B7 or vehicle alone. FIG. 38A shows SDS-PAGEblotted of individual soluble supernatants from 20% brain homogenates ofcontrol infused 19 m.o. 3×Tg mice (Lanes 1-8), and 19 m.o. 3×Tg miceinfused with GW-23B7 (Lanes 9-16). Top left panel Fast Green proteinreversible stain for comparable loading; top right panel immunoblot with4G8/6E10 antibodies specific for Aβ peptides; bottom left panelimmunoblot with PHF-1 antibody specific for hyperphosphorylated tau; andbottom right panel immunoblot with GW-23B7. Different molecular weightoligomeric forms analyzed are identified by color coded arrows, samecolor same molecular weight. FIG. 38B shows densitometric quantitationof the oligomer bands coded with color arrows in FIG. 38A. Statisticalanalysis by two-way ANOVA; *: p<0.05; **: p<0.01.

DETAILED DESCRIPTION OF THE INVENTION

Described is a novel approach to produce conformational monoclonalantibodies selected to specifically react with the β-sheet secondarystructure of pathological proteins, including pathological oligomericconformers that are characteristic of many neurodegenerative diseases.Contrary to past and current efforts, a mammalian non-self-antigen isutilized as an immunogen. The small, non-self peptide selected wascovalently polymerized with glutaraldehyde until it reached a highI3-sheet secondary structure content, and species between 10-100 kDathat are immunogenic, stable and soluble (p13Bri). Inoculation of p13Briin mice elicited antibodies to the peptide and the β-sheet secondarystructure conformation. Hybridomas were produced and clones selected fortheir reactivity with at least two different oligomeric conformers fromAlzheimer's, Parkinson and/or Prion diseases. The resultingconformational monoclonals are able to detect pathological oligomericforms in different human neurodegenerative diseases by ELISA,immunohistochemistry and immunoblots. This technological approach hasresulted in the development of monoclonal antibodies as described hereinthat are useful tools for detection, monitoring and treatment ofmultiple misfolding disorders.

The antibodies and binding fragments thereof that are described hereinselectively recognize and bind the toxic, pathological forms ofamyloidogenic proteins, but not the non-toxic, non-pathological forms ofthese proteins or peptides. Toxic forms of the amyloidogenic proteinsinclude soluble oligomeric forms and insoluble fibrous protein/peptideaggregates of amyloid proteins or peptides. FIG. 1A depicts theconformational equilibrium pathway of several exemplary proteins, i.e.,Aβ, Tau, α-synuclein, and prion, as they progress from soluble,non-toxic proteins to insoluble, toxic proteins. The pathway involves aninitial conformational transition from non-pathological soluble proteinsto modified monomer forms containing high β-sheet secondary structure.Continued transition of the β-sheet containing monomers results in theformation of fibrillogenic β-sheet oligomers and finally insolublefibril deposits. The oligomeric form of the amyloid proteins or peptidesis a multimeric species formed from modified monomers, dimers, trimers,etc. of the protein or peptide. The various conformational forms of theamyloidogenic proteins (i.e., modified monomers→oligomers→fibrils) allcontain a secondary structure that is predominately β-sheet, which isspecifically recognized in the modified monomers and oligomers by theantibodies described herein. The final fibrils are compacted and theβ-sheet secondary structure gets either buried or inaccessible to thehydrophilic solvent soluble antibodies described herein. This β-sheetsecondary structure is absent or is present at a low percentage in thenon-pathological forms of these proteins. Some β-sheet secondarystructures of non-pathological forms of proteins are found within theirinterior; hence, these β-sheets are of difficult access and recognitionby the antibodies described herein.

As used herein, “amyloidogenic protein” encompasses any insolublefibrous protein/peptide aggregate that can be deposited intra- orextracellularly within the body. Amyloidogenic protein/peptidedeposition may be organ-specific (e.g., central nervous system,pancreas, etc.) or systemic. As depicted in FIG. 1A, all amyloidogenicproteins in the oligomeric forms share in common a β-sheet secondarystructure that can be recognized by certain anti-conformationalantibodies and fragments thereof that are described in herein.Amyloidogenic proteins recognized by the antibodies described hereininclude, without limitation, amyloid precursor protein APP, amyloid β,prion and prion proteins, α-synuclein, tau, insulin, ABri precursorprotein, ADan precursor protein, amylin, huntingtin, TDP-43, Doppel,apolipoprotein AI, apolipoprotein AII, lysozyme, cystatin C, gelsolin,protein, atrial natriuretic factor, calcitonin, keratoepithelin,lactoferrin, immunoglobulin light chains, transthyretin, serum amyloid A(SAA) and derived amyloid A (AA), β2-microglobulin, immunoglobulin heavychains, fibrinogen alpha chains, prolactin, keratin, amylin, and medin.Amyloid deposition may occur as its own entity or as a result of anotherillness (e.g., multiple myeloma, chronic infection, or chronicinflammatory disease).

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired binding activity, i.e., binding to the toxic, pathologicaloligomeric forms of amyloidogenic proteins.

In one embodiment, the antibody of the disclosure is an immunoglobulin(Ig) molecule and comprises four polypeptide chains, i.e., two heavy (H)chains and two light (L) chains linked by disulfide bonds. Five types ofmammalian Ig heavy chains are known: α, δ, ε, γ, and μ, wherein the typeof heavy chain defines the class (isotype) of the antibody. Antibodiesof the disclosure can be of any class (e.g., IgG, IgE, IgM, IgD, andIgA), and subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). In oneembodiment, the antibody of the disclosure is an IgM antibody. Inanother embodiment, the antibody disclosed herein is not an IgGantibody.

The heavy chain may contain two regions, the constant region (C_(H)) andthe variable region (V_(H)). The constant region shares high homology inall naturally occurring antibodies of the same isotype within the samespecies. Like the heavy chain, a light chain may also consist of oneconstant domain (C_(L)) and one variable domain (V_(L)). In mammalsthere are two types of immunoglobulin light chain, lambda (λ) and kappa(κ). The unique binding property or antigen binding specificity of agiven antibody is determined by the variable (V) regions. In particular,three hyper-variable loops in each the light (V_(L)) and the heavy(V_(H)) chains, known as complementarity determining regions (CDR), areresponsible for the antigen binding specificity. These regions aredescribed in more detail infra.

An antibody fragment of the disclosure is a molecule containing anantigen binding region or antigen binding domain of a full antibody(e.g., the V_(H) region, the V_(L) region, or a combination of bothregions). In one embodiment, the antibody fragment comprises asingle-chain polypeptide containing one, two, or three of the CDRs ofthe light-chain variable domain, or one, two, or three of the CDRs ofthe heavy chain variable region. In another embodiment, the antibodyfragment of the disclosure is a single domain antibody (also referred toas a nanobody), e.g., a peptide chain of about 110 amino acids longcomprising one heavy chain variable region domain or one light chainvariable region domain of a full antibody. In another embodiment, theantibody fragment is a fragment antigen-binding (F(ab)) fragment or aF(ab′)₂ fragment.

Antibodies and antibody fragments of the present disclosure alsoencompass mutants, variants, or derivatives of the disclosed antibodiesor fragments thereof which retain the essential epitope binding featuresof an Ig molecule. For example, the single domain antibodies can bederived from camelid (V_(HH) domains) or cartilaginous fish (V-NAR)variable domains, alone or fused to an Fc domain. In another embodiment,the antibody fragment comprises the heavy chain and light chain variableregions fused together to form a single-chain variable domain antibody(scFv) or a single-chain variable domain with an Fc portion (i.e., ascFv-Fc, e.g., a minibody.). In another embodiment, the antibodyfragment is a divalent or bivalent single-chain variable fragment,engineered by linking two scFvs together either in tandem (i.e., tandemscFv), or such that they dimerize to form diabodies. In yet anotherembodiment, the antibody is a trivalent single chain variable fragment,engineered by linking three scFvs together, either in tandem or in atrimer formation to form triabodies. In another embodiment, the antibodyis a tetrabody single chain variable fragment. In another embodiment,the antibody is a “linear antibody” which is an antibody comprising apair of tandem Fd segments (V_(H)-C_(H)1-V_(H)-C_(H)1) that form a pairof antigen binding regions (see Zapata et al. Protein Eng.8(10):1057-1062 (1995), which is hereby incorporated by reference in itsentirety).

Antibody and antibody fragments disclosed herein can be mono-valent,bi-valent, or tri-valent with regard to binding domains, and the bindingdomains may be mono-specific, bi-specific, or tri-specific in bindingspecificity by design.

As noted above, the V_(H) and V_(L) regions of an antibody aresubdivided into regions of hypervariability, termed complementarilydetermining regions (CDR). The CDRs are interspersed with regions thatare more conserved in each family of V genes, termed framework regions(FR). These FR regions are specific to place in the proper spatialconfiguration the contact amino acid residues of the CDRs that areresponsible for most of the binding capacity of the antibody. Each V_(H)and V_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4.

The three CDRs in each of the variable regions of the heavy chain andthe light chain are designated CDR1, CDR2 and CDR3 for each of thevariable regions (i.e., (L-CDR1, 2 and 3 of light chain and H-CDR1, 2,and 3 of heavy chain). The term “CDR set” refers to a group of threeCDRs that occur in a single variable region capable of binding theantigen. The exact boundaries of these CDRs have been defineddifferently according to different systems. The system described byKabat (Kabat et al., Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987) and (1991), whichis hereby incorporated by reference in its entirety) not only providesan unambiguous residue numbering system applicable to any variableregion of an antibody, but also provides precise residue boundariesdefining the three CDRs. These CDRs may be referred to as Kabat CDRs.Chothia and coworkers (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987)and Chothia et al., Nature 342:877-883 (1989) which are herebyincorporated by reference in their entirety, describe certainsub-portions within Kabat CDRs that adopt nearly identical peptidebackbone conformations, despite having great diversity at the level ofamino acid sequence. These regions may be referred to as Chothia CDRs,which have boundaries that overlap with Kabat CDRs. Other boundariesdefining CDRs overlapping with the Kabat CDRs have been described byPadlan, FASEB J. 9:133-139 (1995) and MacCallum, J Mol Biol262(5):732-45 (1996), which are hereby incorporated by reference intheir entirety.

In one embodiment, the CDRs and FRs of the heavy and light chainvariable regions of the antibody or fragment thereof of the presentdisclosure are defined in accordance with Kabat et al., “Sequences ofProteins of Immunological interest” 5^(th) ed. (1991), which is herebyincorporated by reference in its entirety. In accordance with the Kabatsystem, an antibody of the present disclosure comprises a heavy chainvariable region where FR1 encompasses residues 1-25 (after the leadersequences) and contains a conserved cysteine (Cys) residue at position22. CDR1 region extends from residue 26 to about residue 35. The residueat position 35 is defined by the conserved tryptophan (Trp) residue atposition 36, which is essential for antibody folding. The CDR1 maycontain up to 2 residue insertions (i.e., 35A and 35B). FR2 of the heavychain variable region begins at the tryptophan residue at about position36 and extends up to the duplet Isoleucine-Glycine (Ile-Gly) at position48-49 in some V_(H) families or Leucine (Leu) at position 48 and Alanine(Ala), Serine (Ser) at position 49 in other V_(H) families. CDR2 of theV_(H) extends from residue 50 to residue 65 with 3 possible insertionsor deletions in the middle of the CDR (typically 16-20 residues inlength). The third framework of the V_(H) extends from the conservedArginine (Arg) or Lysine (Lys) residue at position 66 to position 94,which is two amino acid residues after the consensus Cys residue atposition 92. FR3 may comprise 3 insertions, therefore ranging between29-32 amino acid residues in total. CDR3 starts at residue 95, asdefined by the conserved Cys at position 92, is between 3-25 amino acidresidues in length, and is made by the recombination of three differentgenes, i.e. a VH gene of any family, a partial or complete DH gene, anda JH gene.

In accordance with the Kabat numbering system, the FR1 of the lightchain variable region (V_(L)) of an antibody or fragment thereof asdescribed herein extends from residue 1 (after the leader sequence) tothe conserved Cys at residue 23. CDR1 begins after the conserved Cysresidue, i.e., at position 24 and extends 10-17 residues to the aminoacid residue before the conserved Trp residue at about position 35. TheTrp residue is essential for antibody folding. The second framework ofthe V_(L) begins at the conserved Trp residue and extends to theconserved Tyrosine (Tyr) at position 49. CDR2 of the V_(L) extends fromposition 50 after the conserved Tyr residue to position 56, endingbefore the conserved Gly residue or equivalent at position 57. CDR2typically has seven amino acid residues or less. The third frameworkbegins at the consensus Gly at position 57 and extends to the Cys asposition 88. The cysteine at position 88 forms the disulfide bridge withthe conserved cysteine at position 23. CDR3 of the V_(L) starts atposition 89 (after the consensus Cys at position 88) and extends toposition 97. Residues 97 and 98 are conserved threonine andphenylalanine. The length of CDR3 is made by the recombination of twodifferent genes, i.e. a V_(L) gene of any family and a J_(L) gene. Thus,the length of CDR3 varies as it may contain up to six amino acid residueinsertions. The fourth framework region begins at position 98 andextends through position 107.

In one embodiment, the antibody or binding fragment thereof describedherein is a chimeric antibody. A chimeric antibody is an antibody whereone portion of the amino acid sequence of each of the heavy and lightchains is homologous to corresponding sequences in an antibody derivedfrom a particular species or belonging to a particular class, while theremaining segment of each chain is homologous to corresponding sequencesin another species or class. Typically the variable region of both lightand heavy chains mimics the variable regions of antibodies derived fromone species of mammals, while the constant portions are homologous tosequences of antibodies derived from another. For example, the variableregion can be derived from presently known sources using readilyavailable B-cells or hybridomas from non-human host organisms incombination with constant regions derived from, for example, human cellpreparations. Methods of making chimeric antibodies are well known inthe art, see e.g., U.S. Pat. No. 4,816,567; and Morrison et al.,“Chimeric human antibody molecules: mouse antigen-binding domains withhuman constant region domains” Proc. Natl. Acad. Sci. USA 81:6851-6855(1984), which are hereby incorporated by reference in their entirety).

In another embodiment, the antibody or binding fragment thereof is aCDR-grafted antibody. A “CDR-grafted antibody” is an antibody whichcomprises heavy and light chain variable region sequences of onespecies, where one or more of the CDR regions are replaced with CDRregions of another species. For example, in one embodiment the CDRgrafted antibody comprises human or humanized heavy and light chainvariable regions, where one or more of the CDRs within these regions isreplaced with one or more CDRs from another species, e.g., murine CDRsas shown in Tables 1 and 2 herein.

In another embodiment, the antibody or binding fragment thereof is ahumanized antibody. A humanized antibody is an antibody or a variant,derivative, analog or portion thereof which comprises a framework regionhaving substantially the amino acid sequence of a human antibody and acomplementary determining region having substantially the amino acidsequence of a non-human antibody. As used herein, the term“substantially” in the context of a CDR refers to a CDR having an aminoacid sequence that is at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98% or at least99% identical to the amino acid sequence of a non-human antibody CDR.Likewise, the term “substantially” in the context of a FR refers to a FRhaving an amino acid sequence that is at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98% or at least 99% identical to the amino acid sequence of ahuman FR. A humanized antibody comprises substantially all of at leastone, and typically two, variable domains (Fab, Fab′, F(ab′)₂, Fv) inwhich all or substantially all of the CDR regions correspond to those ofa non-human immunoglobulin (i.e., the donor antibody) and all orsubstantially all of the framework regions are those of a human orhumanized immunoglobulin framework sequence (i.e., the acceptorantibody).

Methods of humanizing antibodies are well known in the art, see e.g.,Almagro and Fransson, “Humanization of Antibodies,” Frontiers inBioscience 13:1619-1633 (2008), U.S. Pat. No. 6,054,297 to Carter etal., U.S. Pat. No. 8,343,489, and U.S. Patent Application PublicationNo. US20100261620 to Almagro et al., which are hereby incorporated byreference in their entirety. The human or humanized framework sequencescan be chosen based on known structure, i.e., a fixed frameworksequence, sequence homology to the framework sequences of the donorantibody (e.g., the antibody from which the CDRs are derived), i.e., abest-fit framework sequence, or a combination of both approaches.Regardless of the method chosen to select the human framework sequence,the sequences can be selected from mature framework sequences, germlinegene sequences, or consensus framework sequences. Compatible humanframework sequences are those that are similar in both length andsequence to the framework sequence of the donor antibody sequence (i.e.,the antibody from which the CDRs are derived) to ensure proper foldingof the antibody and binding domain formation.

In one embodiment, the humanized framework sequence of a humanizedantibody of the disclosure comprises a consensus framework sequence. Aconsensus framework sequence is derived from a consensus immunoglobulinsequence, which is the sequence formed from the most frequentlyoccurring amino acids (or nucleotides) in a family of relatedimmunoglobulin sequences (see e.g., WINNAKER, “From Genes to Clones:Introduction to Gene Technology” (1987); Carter et al., Proc. Natl.Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol., 151:2623(1993), which are hereby incorporated by reference in their entirety).In a family of immunoglobulins, each position in the consensus sequenceis occupied by the amino acid residue occurring most frequently at thatposition in the family. If two amino acids occur equally frequently,either can be included in the consensus sequence.

In another embodiment, a humanized antibody or binding fragment thereofas disclosed herein comprises a fixed framework region. Human heavychain and light chain FR sequences known in the art can be used as heavychain and light chain “acceptor” framework sequences (or simply,“acceptor” sequences) to humanize a non-human antibody using techniquesknown in the art (see e.g., (Sims et al., J. Immunol., 151:2296 (1993);Chothia et al., J. Mol. Biol., 196:901 (1987), which are herebyincorporated by reference in their entirety). In one embodiment, humanheavy chain and light chain acceptor sequences are selected from theframework sequences listed in publicly available databases such asV-base or in the international ImMunoGeneTics® (IMGT®) informationsystem.

Humanized antibodies or binding fragments thereof as described hereinmay also comprise at least a portion of an immunoglobulin constantregion (Fc), typically that of a human immunoglobulin. In oneembodiment, the humanized antibody disclosed herein comprises the lightchain as well as at least the variable domain of a heavy chain. Thehumanized antibody may further comprise the CH1, hinge, CH2, CH3, andCH4 regions of the heavy chain. In another embodiment, the humanizedantibody comprises only a humanized light chain. In another embodiment,the humanized antibody comprises only a humanized heavy chain. Inanother embodiment, the humanized antibody comprises only a humanizedvariable domain of a light chain and/or a humanized variable domain of aheavy chain.

Humanized antibodies and binding fragments thereof as described hereinmay be selected from any class of immunoglobulins, including IgM, IgG,IgD, IgA and IgE, and any isotype, including without limitation IgG1,IgG2, IgG3, IgG4, IgA1, and IgA2. The humanized antibody or bindingfragment thereof may comprise sequences from more than one class orisotype, and particular constant domains may be selected to optimizedesired effector functions using techniques well-known in the art.

In one embodiment, the antibodies and binding fragments thereof asdescribed herein are human antibodies. Methods of producing humanantibodies that are known in the art are suitable for use in accordancewith the present disclosure. For example, one can produce transgenicanimals (e.g., mice) that are capable, upon immunization, of producing afull repertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array into such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl Acad. Sci. USA 90:2551 (1993);Jakobovits et al., Nature 362:255-258 (1993); U.S. Pat. No. 5,545,806 toLonberg et al, U.S. Pat. No. 5,569,825 to Lonberg et al, and U.S. Pat.No. 5,545,807 to Surani et al, which are hereby incorporated byreference in their entirety.

Alternatively, phage display technology (McCafferty et al., Nature348:552-553 (1990), which is hereby incorporated by reference in itsentirety) can be used to produce human antibodies and antibody fragmentsin vitro, from immunoglobulin variable (V) domain gene repertoires fromunimmunized donors. According to this technique, antibody V domain genesare cloned in-frame into either a major or minor coat protein gene of afilamentous bacteriophage and displayed as functional antibody fragmentson the surface of the phage particle. Because the filamentous particlecontains a single-stranded DNA copy of the phage genome, selectionsbased on the functional properties of the antibody also result inselection of the gene encoding the antibody exhibiting those properties.Thus, the phage mimics some of the properties of the B cell. Phagedisplay can be performed in a variety of formats, see e.g., Johnson andChiswell, Current Opinion in Structural Biology 3:564-571 (1993), whichis hereby incorporated by reference in its entirety. A repertoire of Vgenes from unimmunized human donors can be constructed and antibodies toa diverse array of antigens (including self-antigens) can be isolatedessentially following the techniques described by Marks et al., J. Mol.Biol. 222:581-597 (1991), Griffith et al., EMBO J. 12:725-734 (1993),see e.g., U.S. Pat. No. 5,565,332 to Hogenboom and U.S. Pat. No.5,573,905 to Lerner et al., which are hereby incorporated by referencein their entirety.

The antibodies and binding fragments thereof described herein can behuman antibodies or humanized antibodies (fully or partially humanized)as described supra. Alternatively, the antibodies and binding fragmentsthereof can be animal antibodies such as, but not limited to, a bird(for example, a duck, chicken, or a goose), a shark, a whale, or amammal, including a non-primate (for example, a cow, a pig, a camel orall camelids, a llama, a horse, a goat, a rabbit, a sheep, a deer orother cervids, a hamster, a guinea pig, a cat, a dog, a rat, a mouse,etc.) or a non-human primate (for example, a monkey, a chimpanzee,etc.).

Methods of antibody production, in particular, monoclonal antibodyproduction, may be carried out using the methods described herein andthose well-known in the art (MONOCLONAL ANTIBODIES—PRODUCTION,ENGINEERING AND CLINICAL APPLICATIONS (Mary A. Ritter and Heather M.Ladyman eds., 1995), which is hereby incorporated by reference in itsentirety). Generally, the process involves obtaining immune cells(lymphocytes) from the spleen of an animal which has been previouslyimmunized with the antigen of interest (e.g., polymerized Bri peptide asdescribed in the Examples herein) either in vivo or in vitro.

The antibody-secreting lymphocytes are then fused with myeloma cells ortransformed cells, which are capable of replicating indefinitely in cellculture, thereby producing an immortal, immunoglobulin-secreting cellline. Fusion with mammalian myeloma cells or other fusion partnerscapable of replicating indefinitely in cell culture is achieved bystandard and well-known techniques, for example, by using polyethyleneglycol (PEG) or other fusing agents (Milstein and Kohler, “Derivation ofSpecific Antibody-Producing Tissue Culture and Tumor Lines by CellFusion,” Eur J Immunol 6:511 (1976), which is hereby incorporated byreference in its entirety). The immortal cell line, which is preferablymurine, but may also be derived from cells of other mammalian species,is selected to be deficient in enzymes necessary for the utilization ofcertain nutrients, to be capable of rapid growth, and have good fusioncapability. The resulting fused cells, or hybridomas, are cultured, andthe resulting colonies screened for the production of the desiredmonoclonal antibodies. Colonies producing such antibodies are cloned,and grown either in vivo or in vitro to produce large quantities ofantibody.

In another embodiment, monoclonal antibodies can be isolated fromantibody phage libraries generated using the techniques described inMcCafferty et al., “Phage Antibodies: Filamentous Phage DisplayingAntibody Variable Domains,” Nature 348:552-554 (1990), which is herebyincorporated by reference in its entirety. Clackson et al., “MakingAntibody Fragments using Phage Display Libraries,” Nature 352:624-628(1991); and Marks et al., “By-Passing Immunization. Human Antibodiesfrom V-Gene Libraries Displayed on Phage,” J. Mol. Biol. 222:581-597(1991), which are hereby incorporated by reference in their entirety,describe the isolation of murine and human antibodies, respectively,using phage libraries. Subsequent publications describe the productionof high affinity (nM range) human antibodies by chain shuffling (Markset al., BioTechnology 10:779-783 (1992), which is hereby incorporated byreference in its entirety), as well as combinatorial infection and invivo recombination as a strategy for constructing very large phagelibraries (Waterhouse et al., Nuc. Acids. Res. 21:2265-2266 (1993),which is hereby incorporated by reference in its entirety). Thus, thesetechniques are viable alternatives to traditional monoclonal antibodyhybridoma techniques for isolation of monoclonal antibodies.

Alternatively, monoclonal antibodies can be made using recombinant DNAmethods as described in U.S. Pat. No. 4,816,567 to Cabilly et al, whichis hereby incorporated by reference in its entirety. The polynucleotidesencoding a monoclonal antibody are isolated from mature B-cells orhybridoma cells, for example, by RT-PCR using oligonucleotide primersthat specifically amplify the genes encoding the heavy and light chainsof the antibody. The isolated polynucleotides encoding the heavy andlight chains are then cloned into suitable expression vectors, whichwhen transfected into host cells such as E. coli cells, simian COScells, Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, generate monoclonalantibodies.

The polynucleotide(s) encoding a monoclonal antibody can further bemodified using recombinant DNA technology to generate alternativeantibodies. For example, the constant domains of the light and heavychains of a mouse monoclonal antibody can be substituted for thoseregions of a human antibody to generate a chimeric antibody.Alternatively, the constant domains of the light and heavy chains of amouse monoclonal antibody can be substituted for a non-immunoglobulinpolypeptide to generate a fusion antibody. In other embodiments, theconstant regions are truncated or removed to generate the desiredantibody fragment of a monoclonal antibody. Furthermore, site-directedor high-density mutagenesis of the variable region can be used tooptimize specificity and affinity of a monoclonal antibody.

FIG. 1B is a schematic depiction of an exemplary method for antigenpreparation and monoclonal antibody production to generate theantibodies disclosed herein. As described in the Examples herein, a 13mer peptide of ABri, which is a non-self antigen lacking tertiarystructure, is suitable for directional covalent polymerization to forman immunogenic, non-self, stable, and soluble antigen for antibodyproduction. This antigen is produced by a controlled reaction withglutaraldehyde to form predominantly β-sheet oligomers. The oligomers donot progress to form fibrils as shown in the normal equilibrium pathwaythat the 13 mer ABri peptide would undergo. The stable, soluble,non-self oligomer formed as a result of the controlled glutaraldehydereaction is administered to mice or other mammals to induce a polyclonalantibody response (FIG. 1C). The polyclonal antibodies generated uponinoculation primarily recognize either ABri primary sequence or thesecondary β-sheet structure. The spleen of an animal with a goodresponse to β-sheet conformers is dislodged and the splenocytes arefused with the Sp2/0 supplemented Sp2/mIL6 partner to producehybridomas. Hybridomas producing clones that bind to β-sheetconformational structure common to oligomeric forms of Aβ, Tau,α-synuclein, and PrP are positively selected for and expanded. As shownin FIG. 1C, the selectively expanded antibodies recognize the β-sheetsecondary structure that is shared by various amyloidogenic proteins atvarious stages during their transition from non-toxic to toxicpathological forms. While the process in FIG. 1B depicts the utilizationof a 13-mer ABri peptide, various other peptides and fusion peptidesthat are suitable for controlled glutaraldehyde polymerization to forman immunogenic, non-self, stable and soluble antigen with high β-sheetsecondary structure suitable for antibody production are disclosed inU.S. Pat. No. 8,409,584 to Wisniewski et al., which is herebyincorporated by reference in its entirety.

In one embodiment, the antibody or binding fragment thereof as disclosedherein comprises a heavy chain variable region (HCVR) having a H-CDR1with an amino acid sequence selected from SEQ ID NOs: 23-26, and 50, ora modified amino acid sequence thereof containing 1, 2, 3, 4, 5, 6, 7,8, or 9 amino acid residue modifications as compared to SEQ ID NOs:23-26, or 50 that maintain or enhance binding specificity of the H-CDR1.In one embodiment, the amino acid sequence of the H-CDR1 contains nomore than 1, 2, or 3 amino acid modifications as compared to any one ofSEQ ID NOs: 23-26, and 50. The HCVR further comprises a H-CDR2 with anamino acid sequence selected from SEQ ID NOs: 27-30, and 51, or amodified amino acid sequence thereof containing 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16 amino acid residue modifications ascompared to SEQ ID NOs: 27-30, or 51 that maintain or enhance bindingspecificity of the H-CDR2. In one embodiment, the amino acid sequence ofthe H-CDR2 contains no more than 1, 2, 3, or 4 amino acid modificationsas compared to any one of SEQ ID NOs: 27-30, and 51. The HCVR of theantibody or binding fragment thereof comprises a H-CDR3 with an aminoacid sequence selected from SEQ ID NOs: 31-34, and 52, or a modifiedamino acid sequence thereof containing 1, 2, 3, 4, 5, or 6 amino acidresidue modifications as compared to SEQ ID NOs: 31-34, or 52 thatmaintain or enhance binding specificity of the H-CDR3. In oneembodiment, the amino acid sequence of the H-CDR3 contains no more than1, 2, or 3 amino acid modifications as compared to any one of SEQ IDNOs: 31-34, and 52. The amino acid sequences of SEQ ID NOs: 23-34 and50-52 are provided in Table 1 below.

TABLE 1 Exemplary Heavy Chain CDR Amino Acid Sequences SEQ ID SEQ IDSEQ ID Ab Name HCVR CDR1 NO HCVR CDR2 NO HCVR CDR3 NO TF-10E8 GYSFTSYYIH23 WIYPGSGNTKYNEKFKG 27 SYGDYDY 31 FT-12E1 GFSLTSYGVH 24VIWSGGSTDYNAAFIS 28 NPSAYYSNYWFAY 32 WG-3D7 GYSFTGYYMH 25EINPSTGGTSYNQKFKG 29 DYYSKAY 33 TF-10F7 GYSFTSYYIH 23 WIYPGSGNTKYNEKFKG27 SYGDYDY 31 GW-23B7 GFNIKNTYMH 26 RIDPANGNTKYAPKFQG 30 FYAMDY 34FT-11F2 GFSLSTYGMGVG 50 NIWWNDDKYYNSALKS 51 IGWLLAWFAY 52

In one embodiment, the antibody or binding fragment thereof as disclosedherein comprises a light chain variable region (LCVR) that has acomplementarity-determining region 1 (L-CDR1) having an amino acidsequence of any one of SEQ ID NOs: 35-39, and 53, or a modified aminoacid sequence thereof containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aminoacid residue modifications as compared to any one of SEQ ID NO: 35-39,or 53 that maintain or enhance binding specificity of the L-CDR1. In oneembodiment, the amino acid sequence of the L-CDR1 contains no more than1, 2, 3, or 4 amino acid modifications as compared to any one of SEQ IDNOs: 35-39, or 53. The LCVR further comprises a L-CDR2 having an aminoacid sequence of any one of SEQ ID NOs: 40-44, 54, or 112 or a modifiedamino acid sequence thereof containing 1, 2, 3, or 4 amino acid residuemodifications as compared to SEQ ID NO: 40-44, 54, or 112 that maintainor enhance binding specificity of the L-CDR2. In one embodiment, theamino acid sequence of the L-CDR2 contains no more than 1 or 2 aminoacid modifications as compared to any one of SEQ ID NOs: 40-44, 54, or112. The LCVR further comprises a L-CDR3 having an amino acid sequenceof any one of SEQ ID NOs: 45-49, and 55 or a modified amino acidsequence thereof containing 1, 2, 3, 4, 5, 6, 7, or 8 amino acid residuemodifications as compared to SEQ ID NO: 45-49, or 55 that maintain orenhance binding specificity of the L-CDR3. In one embodiment, the aminoacid sequence of the L-CDR3 contains no more than 1 or 2 amino acidmodifications as compared to any one of SEQ ID NOs: 45-49, or 55. Theamino acid sequences of SEQ ID NOs: 35-49, 53-55, and 112 are providedin Table 2 below.

TABLE 2 Exemplary Light Chain CDR Amino Acid Sequences SEQ ID SEQ IDSEQ ID Ab Name LCVR CDR1 NO LCVR CDR2 NO LCVR CDR3 NO TF-10E8RSSQSLVHSNGNTYLH 35 KVSNRFS  40 SQSTHVPRT 45 FT-12E1 KASQYVGTYVA 36SASYRHT  41 QQYSSSPLT 46 WG-3D7 KASQSVSNDVA 37 YASNRYT  42 QQDYSSPYT 47TF-10F7 RASKSVSTSGYSYMH 38 LVSNLES  43 SQSTHVPRT 48 GW-23B7 RASKSINKYLA39 SGSTLQS  44 QQHNEYPWT 49 FT-11F2(1) KSSQSLLNSRTRKNYLA 53 WASTRES  54KQSYNLLT 55 FT-11F2(1) KSSQSLLNSRTRKNYLA 53 WGSTRYS 112 KQSYNLLT 55

Suitable amino acid modifications to the heavy chain CDR sequences ofTable 1 and/or the light chain CDR sequences of Table 2 include, forexample, conservative substitutions or functionally equivalent aminoacid residue substitutions that result in variant CDR sequences havingsimilar or enhanced binding characteristics to those of the CDRsequences of Table 1 and Table 2. Conservative substitutions are thosethat take place within a family of amino acids that are related in theirside chains. Genetically encoded amino acids can be divided into fourfamilies: (1) acidic (aspartate, glutamate); (2) basic (lysine,arginine, histidine); (3) nonpolar (alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan); and (4)uncharged polar (glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine aresometimes classified jointly as aromatic amino acids. Alternatively, theamino acid repertoire can be grouped as (1) acidic (aspartate,glutamate); (2) basic (lysine, arginine histidine), (3) aliphatic(glycine, alanine, valine, leucine, isoleucine, serine, threonine), withserine and threonine optionally grouped separately asaliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine, tryptophan);(5) amide (asparagine, glutamine); and (6) sulfur-containing (cysteineand methionine) (Stryer (ed.), Biochemistry, 2nd ed, WH Freeman and Co.,1981, which is hereby incorporated by reference in its entirety).Non-conservative substitutions can also be made to the heavy chain CDRsequences of Table 1 and the light chain CDR sequences of Table 2.Non-conservative substitutions involve substituting one or more aminoacid residues of the CDR with one or more amino acid residues from adifferent class of amino acids to improve or enhance the bindingproperties of CDR.

The amino acid sequences of the heavy chain variable region CDRs ofTable 1 and/or the light chain variable region CDRs of Table 2 mayfurther comprise one or more internal neutral amino acid insertions ordeletions that do not alter amyloidogenic protein binding. A neutralamino acid insertion or deletion encompasses the insertion or deletionof any amino acid as long as its insertion or deletion does not alterthe binding specificity of the variable domain region. In oneembodiment, the H-CDR3 having an amino acid sequence of any one of SEQID NOs: 31-34, or 52, further contains one or more internal neutralamino acid insertions or deletions that do not alter amyloidogenicprotein binding. In another embodiment, the L-CDR1, having an amino acidsequence of any one of SEQ ID NOs: 35-39, and 53 further contains one ormore internal neutral amino acid insertions or deletions that do notalter amyloidogenic protein binding.

In one embodiment, the antibody or binding fragment thereof has a heavychain variable region with a H-CDR1 having the amino acid sequence ofSEQ ID NO: 23, or a modified amino acid sequence thereof containing 1,2, or more amino acid residue modifications as compared to SEQ ID NO:23; a H-CDR2 having the amino acid sequence of SEQ ID NO: 27, or amodified amino acid sequence thereof containing 1, 2, 3, 4, or moreamino acid residue modifications as compared to SEQ ID NO: 27; and aH-CDR3 comprising the amino acid sequence of SEQ ID NO: 31, or amodified amino acid sequence thereof, said modified amino acid sequencecontaining 1, 2, or more amino acid modifications as compared to SEQ IDNO: 31. In one embodiment, this antibody further comprises a light chainvariable region that comprises a L-CDR1 having the amino acid sequenceof SEQ ID NO: 35, or a modified amino acid sequence thereof containing1, 2, 3, or 4 amino acid residue modifications as compared to SEQ ID NO:35; a L-CDR2 comprising the amino acid sequence of SEQ ID NO: 40, or amodified amino acid sequence thereof containing 1 or 2 amino acidresidue modifications as compared to said SEQ ID NO: 40; and a L-CDR3comprising the amino acid sequence of SEQ ID NO: 45, or a modified aminoacid sequence thereof containing 1 or 2 amino acid residue modificationsas compared to SEQ ID NO: 45. An exemplary monoclonal antibody havingthese heavy chain and light chain variable regions is referred to hereinas the TF-10E8 antibody.

The TF-10E8 antibody comprises a V_(H) chain amino acid sequence of SEQID NO: 2 as shown below. The CDR regions of the V_(H) chain areunderlined, and the framework regions (i.e., FR1-FR4) flanking the CDRsare shown in bold typeface.

SEQ ID NO: 2 QVQLQQSGPELVKPGASVKISCKAS GYSFTSYYIH WVKQRPGQGLEWIG WIYPGSGNTKYNEKFKG KATLTADTSSSTAYMQLSSLTSEDSAVYYCAR SY GDYDY WGQGTTLTVSS

The TF-10E8 antibody comprises a V_(L) chain amino acid sequence of SEQID NO: 4 as shown below. The CDR regions of the V_(L) chain areunderlined, and the framework regions (i.e., FR1-FR4) flanking the CDRsare shown in bold typeface.

SEQ ID NO: 4 DVVMTQTPLSLPVSLGDQASISC RSSQSLVHSNGNTYLH WYLQKPGQSPK LLIYKVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC SQSTHVP RT FGGGTKLEIK

In one embodiment, the antibody or binding fragment thereof as describedherein comprises a V_(H) region having an amino acid sequence of SEQ IDNO: 2 and/or a V_(L) region having an amino acid sequence of SEQ IDNO:4. In one embodiment the antibody is the monoclonal TF-10E8 antibodyhaving a heavy chain amino acid sequence of SEQ ID NO: 57 and a lightchain amino acid sequence of SEQ ID NO: 59 as shown in Table 6 infra.

In another embodiment, the antibody or binding fragment thereofcomprises a V_(H) region having an amino acid sequence that shares atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94% at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% sequence identity to SEQ ID NO: 2, and/or a V_(L) regionhaving an amino acid sequence that shares at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94% at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% sequence identity toSEQ ID NO: 4.

In one embodiment, the antibody or binding fragment thereof of thepresent disclosure comprises a humanized variant of the V_(H) of SEQ IDNO:2 and/or a humanized variant of the V_(L) of SEQ ID NO: 4, where theframework regions are humanized or replaced with human immunoglobulinframework sequences. As noted supra, suitable human or humanizedframework sequences can be chosen based on their known structure, aconsensus sequence, sequence homology to the framework sequences ofdonor antibody (e.g., the framework sequences of SEQ ID NOs: 2 and 4),or a combination of these approaches. The humanized framework regionsare designed to be similar in length and sequence to the parentalframework sequences of SEQ ID NO: 2 and SEQ ID NO: 4, respectively. Inone embodiment, the humanized framework regions share 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity to theframework regions of SEQ ID NO:2 and SEQ ID NO: 4, respectively. Inanother embodiment, the humanized framework regions are 60%, 65%, 70%,75%, 80%, 85%, 90%, 95% or more similar in length to the frameworkregions of SEQ ID NO:2 and SEQ ID NO: 4, respectively. Humanizedvariants of the V_(H) of SEQ ID NO: 2 and the V_(L) of SEQ ID NO: 4share at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95% sequence identity along the entire length of SEQ ID NO: 2 and SEQ IDNO: 4, respectively.

Another aspect of the present disclosure relates to an antibody orbinding portion thereof (e.g., a human antibody) that competes forbinding to a toxic oligomeric form of an amyloidogenic protein with amonoclonal antibody, wherein said monoclonal antibody comprises a heavychain variable region comprising an H-CDR1 of SEQ ID NO: 23, an H-CDR2of SEQ ID NO: 27, and an H-CDR3 of SEQ ID NO: 31 and a light chainvariable region comprising L-CDR1 of SEQ ID NO: 35, L-CDR2 of SEQ ID NO:40, and L-CDR3 of SEQ ID NO: 45. In accordance with this aspect of thedisclosure, a competitive binding assay, such as Bio-LayerInterferometry (BLI) can be utilized to identify an antibody or bindingportion thereof that competes for binding to a toxic amyloidogenicprotein with the enumerated monoclonal antibody. Other competitivebinding assays known in the art can also be utilized to identify acompetitive binding antibody in accordance with this aspect of thedisclosure.

In another embodiment, the antibody or binding fragment thereof has aheavy chain variable region with a H-CDR1 having the amino acid sequenceof SEQ ID NO: 24, or a modified amino acid sequence thereof containing1, 2, or more amino acid residue modifications as compared to SEQ ID NO:24; a H-CDR2 having the amino acid sequence of SEQ ID NO: 28, or amodified amino acid sequence thereof containing 1, 2, 3, 4, or moreamino acid residue modifications as compared to SEQ ID NO: 28; and aH-CDR3 comprising the amino acid sequence of SEQ ID NO: 32, or amodified amino acid sequence thereof, said modified amino acid sequencecontaining 1, 2, 3, 4, 5, 6, or more amino acid modifications ascompared to SEQ ID NO: 32. In one embodiment, this antibody or bindingfragment thereof further comprises a light chain variable region. Thelight chain variable region comprises a L-CDR1 having the amino acidsequence of SEQ ID NO: 36, or a modified amino acid sequence thereofcontaining 1, 2, 3, or 4 amino acid residue modifications as compared toSEQ ID NO: 36; a L-CDR2 comprising the amino acid sequence of SEQ ID NO:41, or a modified amino acid sequence thereof containing 1 or 2 aminoacid residue modifications as compared to said SEQ ID NO: 41; and aL-CDR3 comprising the amino acid sequence of SEQ ID NO: 46, or amodified amino acid sequence thereof containing 1 or 2 amino acidresidue modifications as compared to SEQ ID NO: 46. An exemplarymonoclonal antibody having these heavy chain and light chain variableregion CDRs is referred to herein as the FT-12E1 antibody.

The FT-12E1 antibody comprises a V_(H) chain amino acid sequence of SEQID NO: 6 as shown below. The CDR regions of the V_(H) chain areunderlined, and the framework regions (i.e., FR1-FR4) flanking the CDRsare shown in bold typeface.

SEQ ID NO: 6 QVQLKQSGPGLVPPSQSLSITCTVS GFSLTSYGVH WVRQSPGKGLEWL GVIWSGGSTDYNAAFIS RLSISKDNSKSQVFFKMNSLQADDTAIYYCA R NPSAYYSNYWFAYWGQGTLVTVSA

The FT-12E1 antibody comprises a V_(L) chain amino acid sequence of SEQID NO: 8 as shown below. The CDR regions of the V_(L) chain areunderlined, and the framework regions (i.e., FR1-FR4) flanking the CDRsare shown in bold typeface.

SEQ ID NO: 8 DIVMTQSQNFMSTSVGDRVSVTC KASQYVGTYVA WYQQKPGQSPKAL IYSASYRHT GVPDRFTGSGSGTDFTLTISNVQSEDLADYFC QQYSSS PLT FGSGTKLEIK

In one embodiment, the antibody or binding fragment thereof as describedherein comprises a V_(H) region amino acid sequence of SEQ ID NO: 6and/or a V_(L) region amino acid sequence of SEQ ID NO: 8. In oneembodiment the antibody is the monoclonal FT-12E1 antibody having aheavy chain amino acid sequence of SEQ ID NO: 61 and a light chain aminoacid sequence of SEQ ID NO: 63 as shown in Table 6 infra.

In another embodiment, the antibody or binding fragment thereofcomprises a V_(H) region having an amino acid sequence that shares atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94% at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% sequence identity to SEQ ID NO: 6, and/or a V_(L) regionhaving an amino acid sequence that shares at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94% at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% sequence identity to SEQ ID NO: 8.

In one embodiment, the antibody or binding fragment thereof of thepresent disclosure comprises a humanized variant of the V_(H) of SEQ IDNO: 6 and/or a humanized variant of the V_(L) of SEQ ID NO: 8, where theframework regions are humanized or replaced with human frameworksequences. In one embodiment, the humanized framework regions share 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity tothe framework regions of SEQ ID NO:6 and SEQ ID NO: 8, respectively. Inanother embodiment, the humanized framework regions are 60%, 65%, 70%,75%, 80%, 85%, 90%, 95% or more similar in length to the frameworkregions of SEQ ID NO:6 and SEQ ID NO: 8, respectively. Humanizedvariants of the V_(H) of SEQ ID NO: 6 and the V_(L) of SEQ ID NO: 8share at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95% sequence identify along the entire length of SEQ ID NO: 6 and SEQ IDNO: 8, respectively.

Another aspect of the present disclosure relates to an antibody orbinding portion thereof (e.g., a human antibody) that competes forbinding to a toxic oligomeric form of an amyloidogenic protein with amonoclonal antibody, wherein said monoclonal antibody comprises a heavychain variable region comprising an H-CDR1 of SEQ ID NO: 24, an H-CDR2of SEQ ID NO: 28, and an H-CDR3 of SEQ ID NO: 32 and a light chainvariable region comprising L-CDR1 of SEQ ID NO: 36, L-CDR2 of SEQ ID NO:41, and L-CDR3 of SEQ ID NO: 46. In accordance with this aspect of thedisclosure, a competitive binding assay, such as Bio-LayerInterferometry (BLI) can be utilized to identify an antibody or bindingportion thereof that competes for binding to a toxic amyloidogenicprotein with the enumerated monoclonal antibody. Other competitivebinding assays known in the art can also be utilized to identify acompetitive binding antibody in accordance with this aspect of thedisclosure.

In another embodiment, the antibody or binding fragment thereof has aheavy chain variable region with a H-CDR1 having the amino acid sequenceof SEQ ID NO: 25, or a modified amino acid sequence thereof containing1, 2, or more amino acid residue modifications as compared to SEQ ID NO:25; a H-CDR2 having the amino acid sequence of SEQ ID NO: 29, or amodified amino acid sequence thereof containing 1, 2, 3, 4, or moreamino acid residue modifications as compared to SEQ ID NO: 29; and aH-CDR3 comprising the amino acid sequence of SEQ ID NO: 33, or amodified amino acid sequence thereof, said modified amino acid sequencecontaining 1 or 2 more amino acid modifications as compared to SEQ IDNO: 33. In one embodiment, this antibody or binding fragment thereoffurther comprises a light chain variable region. The light chainvariable region comprises a L-CDR1 having the amino acid sequence of SEQID NO: 37, or a modified amino acid sequence thereof containing 1, 2, 3,or 4 amino acid residue modifications as compared to SEQ ID NO: 37; aL-CDR2 comprising the amino acid sequence of SEQ ID NO: 42, or amodified amino acid sequence thereof containing 1 or 2 amino acidresidue modifications as compared to said SEQ ID NO: 42; and a L-CDR3comprising the amino acid sequence of SEQ ID NO: 47, or a modified aminoacid sequence thereof containing 1 or 2 amino acid residue modificationsas compared to SEQ ID NO: 47 An exemplary monoclonal antibody havingthese heavy chain and light chain variable region CDRs is referred toherein as the WG-3D7 antibody.

The WG-3D7 antibody comprises a V_(H) chain amino acid sequence of SEQID NO: 10 as shown below. The CDR regions of the V_(H) chain areunderlined, and the framework regions (i.e., FR1-FR4) flanking the CDRsare shown in bold typeface.

SEQ ID NO: 10 EVKLQQSGPELVKPGASVKISCKAS GYSFTGYYMH WVKQSSEKSLEWI GEINPSTGGTSYNQKFKG KATLTVDKSSSTAYMQLKSLTSEDSAVYYC AR DYYSKAY WGQGTLVTVSA

The WG-3D7 antibody comprises a V_(L) chain amino acid sequence of SEQID NO: 12 as shown below. The CDR regions of the V_(L) chain areunderlined, and the framework regions (i.e., FR1-FR4) flanking the CDRsare shown in bold typeface.

SEQ ID NO: 12 SIVMTQTPKFLLVSAGDRVTITC KASQSVSNDVA WYQQKPGQSPKLL IYYASNRYT GVPDRFTGSGYGTDFTFTISTVQAEDLAVYFC QQDYSS PYT FGGGTKLEIK

In one embodiment, the antibody or binding fragment thereof as describedherein comprises a V_(H) region having an amino acid sequence of SEQ IDNO: 10 and/or a V_(L) region having an amino acid sequence of SEQ ID NO:12. In one embodiment the antibody is the monoclonal WG-3D7 antibodyhaving a heavy chain amino acid sequence of SEQ ID NO: 65 and a lightchain amino acid sequence of SEQ ID NO: 67 as shown in Table 6 infra.

In another embodiment, the antibody or binding fragment thereofcomprises a V_(H) region having an amino acid sequence that shares atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94% at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% sequence identity to SEQ ID NO: 10, and/or a V_(L) regionhaving an amino acid sequence that shares at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94% at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% sequence identity toSEQ ID NO: 12.

In one embodiment, the antibody or binding fragment thereof of thepresent disclosure comprises a humanized variant of the V_(H) of SEQ IDNO: 10 and/or a humanized variant of the V_(L) of SEQ ID NO: 12, wherethe framework regions are humanized or replaced with human frameworksequences. In one embodiment, the humanized framework regions share 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity tothe framework regions of SEQ ID NO:10 and SEQ ID NO: 12, respectively.In another embodiment, the humanized framework regions are 60%, 65%,70%, 75%, 80%, 85%, 90%, 95% or more similar in length to the frameworkregions of SEQ ID NO: 10 and SEQ ID NO: 12, respectively. Humanizedvariants of the V_(H) of SEQ ID NO: 10 and the V_(L) of SEQ ID NO: 12share at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95% sequence identify along the entire length of SEQ ID NO: 10 and SEQID NO: 12, respectively.

Another aspect of the present disclosure relates to an antibody orbinding portion thereof (e.g., a human antibody) that competes forbinding to a toxic oligomeric form of an amyloidogenic protein with amonoclonal antibody, wherein said monoclonal antibody comprises a heavychain variable region comprising an H-CDR1 of SEQ ID NO: 25, an H-CDR2of SEQ ID NO: 29, and an H-CDR3 of SEQ ID NO: 33, and a light chainvariable region comprising L-CDR1 of SEQ ID NO: 37, L-CDR2 of SEQ ID NO:42, and L-CDR3 of SEQ ID NO: 47. In accordance with this aspect of thedisclosure, a competitive binding assay, such as Bio-LayerInterferometry (BLI) can be utilized to identify an antibody or bindingportion thereof that competes for binding to a toxic amyloidogenicprotein with the enumerated monoclonal antibody. Other competitivebinding assays known in the art can also be utilized to identify acompetitive binding antibody in accordance with this aspect of thedisclosure.

In another embodiment, the antibody or binding fragment thereof has aheavy chain variable region with a H-CDR1 having the amino acid sequenceof SEQ ID NO: 23, or a modified amino acid sequence thereof containing1, 2, or more amino acid residue modifications as compared to SEQ ID NO:23; a H-CDR2 having the amino acid sequence of SEQ ID NO: 27, or amodified amino acid sequence thereof containing 1, 2, 3, 4, or moreamino acid residue modifications as compared to SEQ ID NO:27; and aH-CDR3 comprising the amino acid sequence of SEQ ID NO: 31, or amodified amino acid sequence thereof, said modified amino acid sequencecontaining 1 or 2 more amino acid modifications as compared to SEQ IDNO: 31. In one embodiment, this antibody or binding fragment thereoffurther comprises a light chain variable region. The light chainvariable region comprises a L-CDR1 having the amino acid sequence of SEQID NO: 38, or a modified amino acid sequence thereof containing 1, 2, 3,or 4 amino acid residue modifications as compared to SEQ ID NO: 38; aL-CDR2 comprising the amino acid sequence of SEQ ID NO: 43, or amodified amino acid sequence thereof containing 1 or 2 amino acidresidue modifications as compared to said SEQ ID NO: 43; and a L-CDR3comprising the amino acid sequence of SEQ ID NO: 48, or a modified aminoacid sequence thereof containing 1 or 2 amino acid residue modificationsas compared to SEQ ID NO: 48 An exemplary monoclonal antibody havingthese heavy chain and light chain variable region CDRs is referred toherein as the TF-10F7 antibody.

The TF-10F7 antibody comprises a V_(H) chain amino acid sequence of SEQID NO: 2 as shown below. The CDR regions of the V_(H) chain areunderlined, and the framework regions (i.e., FR1-FR4) flanking the CDRsare shown in bold typeface.

SEQ ID NO: 2 QVQLQQSGPELVKPGASVKISCKAS GYSFTSYYIH WVKQRPGQGLEWI GWIYPGSGNTKYNEKFKG KATLTADTSSSTAYMQLSSLTSEDSAVYYC AR SYGDYDY WGQGTTLTVSS

The TF-10F7 antibody comprises a V_(L) chain amino acid sequence of SEQID NO: 18 as shown below. The CDR regions of the V_(L) chain areunderlined, and the framework regions (i.e., FR1-FR4) flanking the CDRsare shown in bold typeface.

SEQ ID NO: 18 DIVLTQSPASLAVSLGQRATISY RASKSVSISGYSYMH WNQQKPGQP PRLLIYLVSNLES GVPARFSGSGSGTDFTLKISRVEAEDLGVYFC SQ STHVPRT FGGGTKLEIK

In one embodiment, the antibody or binding fragment thereof as describedherein comprises a V_(H) region having an amino acid sequence of SEQ IDNO: 2 and/or a V_(L) region having an amino acid sequence of SEQ ID NO:18. In one embodiment, the antibody is the monoclonal TF-10F7 antibodyhaving a heavy chain amino acid sequence of SEQ ID NO: 69 and a lightchain amino acid sequence of SEQ ID NO: 71 as shown in Table 6 infra.

In another embodiment, the antibody or binding fragment thereofcomprises a V_(H) region having an amino acid sequence that shares atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94% at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% sequence identity to SEQ ID NO: 2, and/or a V_(L) regionhaving an amino acid sequence that shares at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94% at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% sequence identity toSEQ ID NO: 18.

In one embodiment, the antibody or binding fragment thereof of thepresent disclosure comprises a humanized variant of the V_(H) of SEQ IDNO: 2 and/or a humanized variant of the V_(L) of SEQ ID NO: 18, wherethe framework regions are humanized or replaced with human frameworksequences. In one embodiment, the humanized framework regions share 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity tothe framework regions of SEQ ID NO: 2 and SEQ ID NO: 18, respectively.In another embodiment, the humanized framework regions are 60%, 65%,70%, 75%, 80%, 85%, 90%, 95% or more similar in length to the frameworkregions of SEQ ID NO: 2 and SEQ ID NO: 18, respectively. Humanizedvariants of the V_(H) of SEQ ID NO: 2 and the V_(L) of SEQ ID NO: 18share at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95% sequence identify along the entire length of SEQ ID NO: 2 and SEQ IDNO: 18, respectively.

Another aspect of the present disclosure relates to an antibody orbinding portion thereof (e.g., a human antibody) that competes forbinding to a toxic oligomeric form of an amyloidogenic protein with amonoclonal antibody, wherein said monoclonal antibody comprises a heavychain variable region comprising an H-CDR1 of SEQ ID NO: 23, an H-CDR2of SEQ ID NO: 27, and an H-CDR3 of SEQ ID NO: 31, and a light chainvariable region comprising L-CDR1 of SEQ ID NO: 38, L-CDR2 of SEQ ID NO:43, and L-CDR3 of SEQ ID NO: 48. In accordance with this aspect of thedisclosure, a competitive binding assay, such as Bio-LayerInterferometry (BLI) can be utilized to identify an antibody or bindingportion thereof that competes for binding to a toxic amyloidogenicprotein with the enumerated monoclonal antibody. Other competitivebinding assays known in the art can also be utilized to identify acompetitive binding antibody in accordance with this aspect of thedisclosure.

In another embodiment, the antibody or binding fragment thereof has aheavy chain variable region with a H-CDR1 having the amino acid sequenceof SEQ ID NO: 26 or a modified amino acid sequence thereof containing 1,2, or more amino acid residue modifications as compared to SEQ ID NO:26; a H-CDR2 having the amino acid sequence of SEQ ID NO: 30, or amodified amino acid sequence thereof containing 1, 2, 3, 4, or moreamino acid residue modifications as compared to SEQ ID NO:30; and aH-CDR3 comprising the amino acid sequence of SEQ ID NO: 34, or amodified amino acid sequence thereof, said modified amino acid sequencecontaining 1 or 2 more amino acid modifications as compared to SEQ IDNO: 34. In one embodiment, this antibody or binding fragment thereoffurther comprises a light chain variable region. The light chainvariable region comprises a L-CDR1 having the amino acid sequence of SEQID NO: 39, or a modified amino acid sequence thereof containing 1, 2, 3,or 4 amino acid residue modifications as compared to SEQ ID NO: 39; aL-CDR2 comprising the amino acid sequence of SEQ ID NO: 44, or amodified amino acid sequence thereof containing 1 or 2 amino acidresidue modifications as compared to said SEQ ID NO: 44; and a L-CDR3comprising the amino acid sequence of SEQ ID NO: 49, or a modified aminoacid sequence thereof containing 1 or 2 amino acid residue modificationsas compared to SEQ ID NO: 49 An exemplary monoclonal antibody havingthese heavy chain and light chain variable region CDRs is referred toherein as the GW-23B7 antibody.

The GW-23B7 antibody comprises a V_(H) chain amino acid sequence of SEQID NO: 20 as shown below. The CDR regions of the V_(H) chain areunderlined, and the framework regions (i.e., FR1-FR4) flanking the CDRsare shown in bold typeface.

SEQ ID NO: 20 EVQLQQSVAELVRPGASVKLSCTAS GFNIKNTYMH WVKQRPEQGLEWI GRIDPANGNTKYAPKFQG KATITADTSSNTAYLQLSSLTSEDTAIYYC ARGS FYAMDY WGQGTSVTVSS

The GW-23B7 antibody comprises a V_(L) chain amino acid sequence of SEQID NO: 22 as shown below. The CDR regions of the V_(L) chain areunderlined, and the framework regions (i.e., FR1-FR4) flanking the CDRsare shown in bold typeface.

SEQ ID NO: 22 DVQITQSPSYLAASPGETITINCRASKSINKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYHCQQHNEY PWTFGGGTKLEIK

In one embodiment, the antibody or binding fragment thereof as describedherein comprises a V_(H) region having an amino acid sequence of SEQ IDNO: 20 and/or a V_(L) region having an amino acid sequence of SEQ ID NO:22. In one embodiment, the antibody is the monoclonal GW-23B7 antibodyhaving a heavy chain amino acid sequence of SEQ ID NO: 73 and a lightchain amino acid sequence of SEQ ID NO: 75 as shown in Table 6 infra.

In another embodiment, the antibody or binding fragment thereofcomprises a V_(H) region having an amino acid sequence that shares atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94% at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% sequence identity to SEQ ID NO: 20, and/or a V_(L) regionhaving an amino acid sequence that shares at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94% at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% sequence identity toSEQ ID NO: 22.

In one embodiment, the antibody or binding fragment thereof of thepresent disclosure comprises a humanized variant of the V_(H) of SEQ IDNO: 20 and/or a humanized variant of the V_(L) of SEQ ID NO: 22, wherethe framework regions are humanized or replaced with human frameworksequences. In one embodiment, the humanized framework regions share 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity tothe framework regions of SEQ ID NO: 20 and SEQ ID NO: 22, respectively.In another embodiment, the humanized framework regions are 60%, 65%,70%, 75%, 80%, 85%, 90%, 95% or more similar in length to the frameworkregions of SEQ ID NO: 20 and SEQ ID NO: 22, respectively. Humanizedvariants of the V_(H) of SEQ ID NO: 20 and the V_(L) of SEQ ID NO: 22share at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95% sequence identify along the entire length of SEQ ID NO: 20 and SEQID NO: 22, respectively.

Another aspect of the present disclosure relates to an antibody orbinding portion thereof (e.g., a human antibody) that competes forbinding to a toxic oligomeric form of an amyloidogenic protein with amonoclonal antibody, wherein said monoclonal antibody comprises a heavychain variable region comprising an H-CDR1 of SEQ ID NO: 26, an H-CDR2of SEQ ID NO: 30, and an H-CDR3 of SEQ ID NO: 34, and a light chainvariable region comprising L-CDR1 of SEQ ID NO: 39, L-CDR2 of SEQ ID NO:44, and L-CDR3 of SEQ ID NO: 49. In accordance with this aspect of thedisclosure, a competitive binding assay, such as Bio-LayerInterferometry (BLI) can be utilized to identify an antibody or bindingportion thereof that competes for binding to a toxic amyloidogenicprotein with the enumerated monoclonal antibody. Other competitivebinding assays known in the art can also be utilized to identify acompetitive binding antibody in accordance with this aspect of thedisclosure.

In one embodiment, the antibody or binding fragment thereof has a heavychain variable region with a H-CDR1 having the amino acid sequence ofSEQ ID NO: 50, or a modified amino acid sequence thereof containing 1,2, 3, or more amino acid residue modifications as compared to SEQ ID NO:50; a H-CDR2 having the amino acid sequence of SEQ ID NO: 51, or amodified amino acid sequence thereof containing 1, 2, 3, 4, or moreamino acid residue modifications as compared to SEQ ID NO: 51; and aH-CDR3 comprising the amino acid sequence of SEQ ID NO: 52, or amodified amino acid sequence thereof, said modified amino acid sequencecontaining 1, 2, or more amino acid modifications as compared to SEQ IDNO: 52. In one embodiment, this antibody further comprises a light chainvariable region that comprises a L-CDR1 having the amino acid sequenceof SEQ ID NO: 53, or a modified amino acid sequence thereof containing1, 2, 3, or 4 amino acid residue modifications as compared to SEQ ID NO:53; a L-CDR2 comprising the amino acid sequence of SEQ ID NO: 54, or amodified amino acid sequence thereof containing 1 or 2 amino acidresidue modifications as compared to said SEQ ID NO: 54; and a L-CDR3comprising the amino acid sequence of SEQ ID NO: 55, or a modified aminoacid sequence thereof containing 1 or 2 amino acid residue modificationsas compared to SEQ ID NO: 55. In another embodiment, this antibodycomprises a light chain variable region that comprises a L-CDR1 havingthe amino acid sequence of SEQ ID NO: 53, or a modified amino acidsequence thereof containing 1, 2, 3, or 4 amino acid residuemodifications as compared to SEQ ID NO: 53; a L-CDR2 comprising theamino acid sequence of SEQ ID NO: 112, or a modified amino acid sequencethereof containing 1 or 2 amino acid residue modifications as comparedto said SEQ ID NO: 112; and a L-CDR3 comprising the amino acid sequenceof SEQ ID NO: 55, or a modified amino acid sequence thereof containing 1or 2 amino acid residue modifications as compared to SEQ ID NO: 55. Anexemplary monoclonal antibody having these heavy chain and light chainvariable regions is referred to herein as the FT-11F2 antibody.

The FT-11F2 antibody comprises a V_(H) chain amino acid sequence of SEQID NO: 14 as shown below. The CDR regions of the V_(H) chain areunderlined, and the framework regions (i.e., FR1-FR4) flanking the CDRsare shown in bold typeface.

SEQ ID NO: 14 QVTLKESGPGILQPSQTLSLTCSFS GFSLSTYGMGVG WIRQPSGKGLEWL ANIWWNDDKYYNSALKSRLTISKDTSNNQVFLKISSVDTADTATYYCAQ I GWLLAWFAY WGQGTLVTVSA

The FT-11F2 antibody comprises a V_(L) chain amino acid sequence of SEQID NO: 16 as shown below. The CDR regions of the V_(L) chain areunderlined, and the framework regions (i.e., FR1-FR4) flanking the CDRsare shown in bold typeface.

SEQ ID NO: 16 DIVMSQSPSSLAVSAGEKVTMSC KSSQSLLNSRTRKNYLA WYQQKPGQSP KLLIYWASTRES GVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC KQSYNL LT FGAGTKLELK

In another embodiment, the FT-11F2 antibody comprises a VL chain aminoacid sequence of SEQ ID NO: 109 as shown below. The CDR regions of theV_(L) chain are underlined, and the framework regions (i.e., FR1-FR4)flanking the CDRs are shown in bold typeface.

SEQ ID NO: 109 DIVMSQSPSSLAVSAGDKVTMSC KSSQSLLNSRTRKNYLA WYQQKPGQSPKVLVY WGSTRYS GVPDRFTGSGSGTDYTLTVSSVQAEDLAVYFC KQSYNL LT FGAGTKL

In one embodiment, the antibody or binding fragment thereof as describedherein comprises a V_(H) region having an amino acid sequence of SEQ IDNO: 14 and/or a V_(L) region having an amino acid sequence of SEQ IDNO:16. In another embodiment, the antibody or binding fragment thereofas described herein comprises a V_(H) region having an amino acidsequence of SEQ ID NO: 14 and/or a V_(L) region having an amino acidsequence of SEQ ID NO:109.

In another embodiment, the antibody or binding fragment thereofcomprises a V_(H) region having an amino acid sequence that shares atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94% at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% sequence identity to SEQ ID NO: 14, and/or a V_(L) regionhaving an amino acid sequence that shares at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94% at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% sequence identity toSEQ ID NO: 16 or SEQ ID NO: 109.

In one embodiment, the antibody or binding fragment thereof of thepresent disclosure comprises a humanized variant of the V_(H) of SEQ IDNO:14 and/or a humanized variant of the V_(L) of SEQ ID NO: 16 or SEQ IDNO: 109, where the framework regions are humanized or replaced withhuman immunoglobulin framework sequences. As noted supra, suitable humanor humanized framework sequences can be chosen based on their knownstructure, a consensus sequence, sequence homology to the frameworksequences of donor antibody (e.g., the framework sequences of SEQ IDNOs: 14, 16, and 109), or a combination of these approaches. Thehumanized framework regions are designed to be similar in length andsequence to the parental framework sequences of SEQ ID NO: 14, SEQ IDNO: 16, and SEQ ID NO: 109, respectively. In one embodiment, thehumanized framework regions share 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95% or more sequence identity to the framework regions of SEQID NO:14, SEQ ID NO: 16, and SEQ ID NO: 109, respectively. In anotherembodiment, the humanized framework regions are 60%, 65%, 70%, 75%, 80%,85%, 90%, 95% or more similar in length to the framework regions of SEQID NO:14, SEQ ID NO: 16, and SEQ ID NO: 109, respectively. Humanizedvariants of the V_(H) of SEQ ID NO: 14 and the V_(L) of SEQ ID NO: 16 orSEQ ID NO: 109 share at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95% sequence identity along the entire length of SEQ IDNO: 14, SEQ ID NO: 16, and SEQ ID NO: 109, respectively.

Another aspect of the present disclosure relates to an antibody orbinding portion thereof (e.g., a human antibody) that competes forbinding to a toxic oligomeric form of an amyloidogenic protein with amonoclonal antibody, wherein said monoclonal antibody comprises a heavychain variable region comprising an H-CDR1 of SEQ ID NO: 50, an H-CDR2of SEQ ID NO: 51, and an H-CDR3 of SEQ ID NO: 52, and a light chainvariable region comprising L-CDR1 of SEQ ID NO: 53, L-CDR2 of SEQ ID NO:54, and L-CDR3 of SEQ ID NO: 55. In accordance with this aspect of thedisclosure, a competitive binding assay, such as Bio-LayerInterferometry (BLI) can be utilized to identify an antibody or bindingportion thereof that competes for binding to a toxic amyloidogenicprotein with the enumerated monoclonal antibody. Other competitivebinding assays known in the art can also be utilized to identify acompetitive binding antibody in accordance with this aspect of thedisclosure.

In one embodiment, the antibody or binding fragment thereof does notcomprise or consist of the amino acid sequence of mAb 3D6 as disclosedin U.S. Pat. No. 8,409,584 to Wisniewski and Goni.

Another aspect of the present disclosure is directed to an antibodymimetic that binds toxic oligomeric forms of amyloidogenic proteins. An“antibody mimetic” as referred to herein encompasses any organiccompound, e.g., a peptide or polypeptide, that can specifically bind anantigen like an antibody, and is about 3-20 kDa. In one embodiment, theantibody mimetic comprises a scaffold which binds its antigen via aminoacids in exposed loops similar to the CDR loops of an antibody. Theseantibody mimetics include, without limitation, adnectins, lipocalins,Kunitz domain-based binders, avimers, knottins, fynomers, atrimers, andcytotoxic T-lymphocyte associated protein-4 (CTLA4)-based binders(reviewed in Weidle et al., “The Emerging Role of New ProteinScaffold-based Agents for the Treatment of Cancer,” Cancer Genomics &Proteomics 10:155-168 (2013), which is hereby incorporated by referencein its entirety. In accordance with this aspect of the presentdisclosure, the loop binding regions of the antibody mimetic are adaptedto comprise one or more of the heavy chain and/or light chain CDRs ofthe antibodies disclosed herein. For example, an antibody mimetic of thepresent disclosure may comprise a first loop region having an amino acidsequence of any one of SEQ ID NOs: 23-26, and 50, or a modified aminoacid sequence of any one of SEQ ID NOs: 23-26, and 50, said modifiedsequence containing 1 or 2 amino acid residue modifications as comparedto any one of SEQ ID NOs: 23-26, or 50. The antibody mimetic maycomprise another loop region having an amino acid sequence of any one ofSEQ ID NOs: 27-30, and 51, or a modified amino acid sequence of any oneof SEQ ID NOs: 27-30, and 51, said modified sequences containing 1, 2,3, or 4 amino acid residue modifications as compared to any one of SEQID NOs: 27-30, or 51. The antibody mimetic may comprise another loopregion having an amino acid sequence of any one of SEQ ID NOs: 31-34,and 52, or a modified amino acid sequence of any one of SEQ ID NO:31-34, and 52, said modified sequence containing 1, 2, or 3 amino acidresidue modifications as compared to any one of SEQ ID NOs: 31-34, and52. The antibody mimetic may further comprise another loop region havingan amino acid sequence of any one of SEQ ID NOs: 35-39, and 53, or amodified amino acid sequence of any one of SEQ ID NO: 35-39, and 53,said modified sequence containing 1, 2, 3, or 4 amino acid residuemodifications as compared to any one of SEQ ID NO: 35-39, or 53. Theantibody mimetic may comprise another loop region having an amino acidsequence of any one of SEQ ID NOs: 40-44, and 54, or a modified aminoacid sequence of any one of SEQ ID NO: 40-44, and 54, said modifiedsequence containing 1 or 2 amino acid residue modifications as comparedto SEQ ID NO: 40-44, or 54. The antibody mimetic may comprise anotherloop region having an amino acid sequence of any one of SEQ ID NOs:45-49, and 55, or a modified amino acid sequence of any one of SEQ IDNO: 45-49, and 55, said modified sequence containing 1 or 2 amino acidresidue modifications as compared to SEQ ID NO: 45-49, or 55.

In one embodiment, the antibody mimetic comprises one or more modifiedfibronectin type III (FN3) domains (e.g., an adnectin or centyrinmolecule), where each modified FN3 domain has one or more loop regionsthat comprise one or more CDR sequences or modified CDR sequences asdisclosed herein (i.e., the sequences disclosed supra in Tables 1 and2).

The FN3 domain is an evolutionary conserved protein domain that is about100 amino acids in length and possesses a beta sandwich structure. Thebeta sandwich structure of human FN3 comprises seven beta-strands,referred to as strands A, B, C, D, E, F, G, with six connecting loops,referred to as loops AB, BC, CD, DE, EF, and FG that exhibit structuralhomology to immunoglobulin binding domains. Three of the six loops,i.e., loops DE, BC, and FG, correspond topologically to thecomplementarity determining regions of an antibody, i.e., CDR1, CDR2,and CDR3. The remaining three loops are surface exposed in a mannersimilar to antibody CDR3. In accordance with the present disclosure, oneor more of the loop regions of each FN3 domain of the binding moleculeare modified to comprise one or more CDR sequences disclosed herein.

The modified FN3 domain can be a FN3 domain derived from any of the widevariety of animal, yeast, plant, and bacterial extracellular proteinscontaining these domains. In one embodiment, the FN3 domain is derivedfrom a mammalian FN3 domain. Exemplary FN3 domains include, for exampleand without limitation, any one of the 15 different FN3 domains presentin human tenascin C, or the 15 different FN3 domains present in humanfibronectin (FN) (e.g., the 10^(th) fibronectin type III domain).Exemplary FN3 domains also include non-natural synthetic FN3 domains,such as those described in U.S. Pat. Publ. No. 2010/0216708 to Jacobs etal., which is hereby incorporated by reference in its entirety.Individual FN3 domains are referred to by domain number and proteinname, e.g., the 3^(rd) FN3 domain of tenascin (TN3), or the 10^(th) FN3domain of fibronectin (FN10).

Another aspect of the present disclosure is directed to isolatedpolynucleotides encoding the antibody or binding fragment thereof orantibody mimetic as described herein. The nucleic acid moleculesdescribed herein include isolated polynucleotides, portions ofexpression vectors or portions of linear DNA sequences, including linearDNA sequences used for in vitro transcription/translation, and vectorscompatible with prokaryotic, eukaryotic or filamentous phage expression,secretion, and/or display of the antibodies or binding fragments thereofdescribed herein.

In one embodiment, the isolated polynucleotide encodes the heavy chainvariable region of the antibody or binding fragment disclosed herein.This isolated polynucleotide comprises a nucleotide sequence encodingthe H-CDR1 that is selected from any one of the nucleotide sequences ofSEQ ID NOs: 76-80, or a nucleotide sequence comprising 80% sequencesimilarity to any one of SEQ ID NOs: 76-80. The isolated polynucleotidefurther comprises a nucleotide sequence encoding the H-CDR2 that isselected from any one of the nucleotide sequences of SEQ ID NOs: 81-85,or a nucleotide sequence comprising 80% sequence similarity to any oneof SEQ ID NOs: 81-85. The isolated polynucleotide further comprises anucleotide sequence encoding the H-CDR3 that is selected from any one ofthe nucleotide sequences of SEQ ID NOs: 86-90, or a nucleotide sequencecomprising 80% sequence similarity to any one of SEQ ID NOs: 86-90. Thenucleotide sequences encoding the heavy chain CDRs of the antibodies andbinding fragments thereof as described herein are provided in Table 3below.

TABLE 3  Nucleotide Sequences Encoding Antibody Heavy Chain CDRs SEQ SEQSEQ Ab ID ID ID Name HCVR CDR1 NO HCVR CDR2 NO HCVR CDR3 NO TF-10E8GGCTACAGCTTC 76 TGGATTTATCCTGG 81 AGCTATGGTGACTAC 86 ACAAGCTACTATAAGTGGTAATACTA GACTAC ATACACT AGTACAATGAGAAG TTCAAGGGC FT-12E1GGTTTCTCATTA 77 GTGATATGGAGTGG 82 AATCCCTCCGCCTAC 87 ACTAGCTATGGTTGGAAGCACAGACT TATAGTAACTACTGG GTACAC ACAATGCAGCTTTC TTTGCTTAC ATATCCWG-3D7 GGTTACTCATTC 78 GAGATTAATCCTAG 83 GACTACTATAGTAAG 88 ACTGGCTACTACCACTGGTGGTACTA GCTTAC ATGCAC GCTACAACCAGAAG TTCAAGGGC TF-10F7GGCTACAGCTTC 76 TGGATTTATCCTGG 81 AGCTATGGTGACTAC 86 ACAAGCTACTATAAGTGGTAATACTA GACTAC ATACAC AGTACAATGAGAAG TTCAAGGGC GW- GGCTTCAACATT79 AGGATTGATCCTGC 84 TTTTATGCTATGGAC 89 23B7 AAAAACACCTAT GAATGGTAATACTATAC ATGCAC AATATGCCCCGAAG TTCCAGGGC FT-11F2 ACTTATGGTATG 80AACATTTGGTGGAA 85 ATAGGGTGGTTACTA 90 GGTGTAGGT TGATGATAAGTACTGCCTGGTTTGCTTAC ATAACTCAGCCCTG AAGAGC

In one embodiment, the isolated polynucleotide of the disclosurecomprises the nucleotide sequences of SEQ ID NOs: 76, 81, and 86, ornucleotide sequences comprising at least 80%, at least 85%, at least90%, at least 95%, at least 96%, 97%, 98%, or 99% sequence similarity tothe nucleotide sequences of SEQ ID NOs: 76, 81, and 86. An exemplarypolynucleotide comprising the nucleotide sequences of SEQ ID NOs: 76,81, and 86 comprises the nucleotide sequence of SEQ ID NO: 1 (Table 5),which encodes the heavy chain variable region of the TF-10E8 antibodyand the TF-10F7 antibody as disclosed herein. In one embodiment, theisolated polynucleotide encoding the heavy chain variable region of theantibody or fragment thereof of the present disclosure comprises anucleotide sequence that has at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, 97%, 98%, or 99% sequence similarity to thenucleotide sequence of SEQ ID NO: 1.

In another embodiment, the isolated polynucleotide of the disclosurecomprises the nucleotide sequences of SEQ ID NOs: 77, 82, and 87, ornucleotide sequences comprising at least 80%, at least 85%, at least90%, at least 95%, at least 96%, 97%, 98%, or 99% sequence similarity tothe nucleotide sequences of SEQ ID NOs: 77, 82, and 87. An exemplarypolynucleotide comprising the nucleotide sequences of SEQ ID NOs: 77,82, and 87 comprises the nucleotide sequence of SEQ ID NO: 5 (Table 5),which encodes the heavy chain variable region of the FT-12E1 antibody asdisclosed herein. In one embodiment, the isolated polynucleotideencoding the heavy chain variable region of the antibody or fragmentthereof of the present disclosure comprises a nucleotide sequence thathas at least 80%, at least 85%, at least 90%, at least 95%, at least96%, 97%, 98%, or 99% sequence similarity to the nucleotide sequence ofSEQ ID NO: 5.

In another embodiment, the isolated polynucleotide of the disclosurecomprises the nucleotide sequences of SEQ ID NOs: 78, 83, and 88, ornucleotide sequences comprising at least 80%, at least 85%, at least90%, at least 95%, at least 96%, 97%, 98%, or 99% sequence similarity tothe nucleotide sequences of SEQ ID NOs: 78, 83, and 88. An exemplarypolynucleotide comprising the nucleotide sequences of SEQ ID NOs: 78,83, and 88 comprises the nucleotide sequence of SEQ ID NO: 9 (Table 5),which encodes the heavy chain variable region of the WG-3D7 antibody asdisclosed herein. In one embodiment, the isolated polynucleotideencoding the heavy chain variable region of the antibody or fragmentthereof of the present disclosure comprises a nucleotide sequence thathas at least 80%, at least 85%, at least 90%, at least 95%, at least96%, 97%, 98%, or 99% sequence similarity to the nucleotide sequence ofSEQ ID NO: 9.

In another embodiment, the isolated polynucleotide of the disclosurecomprises the nucleotide sequences of SEQ ID NOs: 79, 84, and 89, ornucleotide sequences comprising at least 80%, at least 85%, at least90%, at least 95%, at least 96%, 97%, 98%, or 99% sequence similarity tothe nucleotide sequences of SEQ ID NOs: 79, 84, and 89. An exemplarypolynucleotide comprising the nucleotide sequences of SEQ ID NOs: 79,84, and 89 comprises the nucleotide sequence of SEQ ID NO: 19 (Table 5),which encodes the heavy chain variable region of the GW-23B7 antibody asdisclosed herein. In one embodiment, the isolated polynucleotideencoding the heavy chain variable region of the antibody or fragmentthereof of the present disclosure comprises a nucleotide sequence thathas at least 80%, at least 85%, at least 90%, at least 95%, at least96%, 97%, 98%, or 99% sequence similarity to the nucleotide sequence ofSEQ ID NO: 19.

In another embodiment, the isolated polynucleotide of the disclosurecomprises the nucleotide sequences of SEQ ID NOs: 80, 85, and 90, ornucleotide sequences comprising at least 80%, at least 85%, at least90%, at least 95%, at least 96%, 97%, 98%, or 99% sequence similarity tothe nucleotide sequences of SEQ ID NOs: 80, 85, and 90. An exemplarypolynucleotide comprising the nucleotide sequences of SEQ ID NOs: 80,85, and 90 comprises the nucleotide sequence of SEQ ID NO: 13 (Table 5),which encodes the heavy chain variable region of the FT-11F2 antibody asdisclosed herein. In one embodiment, the isolated polynucleotideencoding the heavy chain variable region of the antibody or fragmentthereof of the present disclosure comprises a nucleotide sequence thathas at least 80%, at least 85%, at least 90%, at least 95%, at least96%, 97%, 98%, or 99% sequence similarity to the nucleotide sequence ofSEQ ID NO: 13.

In one embodiment, the isolated polynucleotide encodes the light chainvariable region of the antibody or binding fragment. This isolatedpolynucleotide comprises a nucleotide sequence encoding the L-CDR1 thatis selected from any one of the nucleotide sequences of SEQ ID NOs:91-96, or a nucleotide sequence comprising at least 80% sequencesimilarity to any one of SEQ ID NOs: 91-96. The isolated polynucleotidefurther comprises a nucleotide sequence encoding the L-CDR2 that isselected from any one of the nucleotide sequences of SEQ ID NOs: 97-102,or a nucleotide sequence comprising at least 80% sequence similarity toany one of SEQ ID NOs: 97-102. The isolated polynucleotide furthercomprises a nucleotide sequence encoding the L-CDR3 that is selectedfrom any one of the nucleotide sequences of SEQ ID NOs: 103-108, or anucleotide sequence comprising at least 80% sequence similarity to anyone of SEQ ID NOs: 103-108. The nucleotide sequences encoding the lightchain CDRs of the antibodies and binding fragments thereof as describedherein are provided in Table 4 below.

TABLE 4  Nucleotide Sequences Encoding Antibody Light Chain CDRs SEQ SEQSEQ ID ID ID Ab Name LCVR CDR1 NO LCVR CDR2 NO LCVR CDR3 NO TF-10E8AGATCTAGTCAGAGCCT 91 AAAGTTTCCAAC 97 TCTCAAAGTACA 103 TGTACACAGTAATGGAACGATTTTCT CATGTTCCTCGG ACACCTATTTACA ACG FT-12E1 AAGGCCAGTCAGTATGT 92TCGGCATCCTAC 98 CAGCAATATAGC 104 GGGTACTTATGTAGCC CGGCATACT AGCTCTCCTCTCACG WG-3D7 AAGGCCAGTCAGAGTGT 93 TATGCATCCAAT 99 CAGCAGGATTAT 105GAGTAATGATGTAGCT CGCTACACT AGCTCTCCGTAC ACG TF-10F7 AGGGCCAGCAAAAGTGT 94CTTGTATCCAAC 100 TCTCAAAGTACA 106 CAGTACATCTGGCTATA CTAGAATCTCATGTTCCTCGG GTTATATGCAC ACG GW- AGGGCAAGTAAGAGCAT 95 TCTGGATCCACC 101CAACAGCATAAT 107 23137 TAACAAATATTTAGCC TTGCAATCT GAATACCCGTGG ACGFT-11F2 AAATCCAGTCAGAGTCT 96 TGGGCATCCACT 102 AAGCAATCTTAT 108GCTCAACAGTAGAACCC AGGGAATCT AATCTGCTCACG GAAAGAACTACT TGGCT

In one embodiment, the isolated polynucleotide of the disclosurecomprises the nucleotide sequences of SEQ ID NOs: 91, 97, and 103, ornucleotide sequences comprising at least 80%, at least 85%, at least90%, at least 95%, at least 96%, 97%, 98%, or 99% sequence similarity tothe nucleotide sequences of SEQ ID NOs: 91, 97, and 103. An exemplarypolynucleotide comprising the nucleotide sequences of SEQ ID NOs: 91,97, and 103 comprises the nucleotide sequence of SEQ ID NO: 3 (Table 5),which encodes the light chain variable region of the TF-10E8 antibody asdisclosed herein. In one embodiment, the isolated polynucleotideencoding the light chain variable region of the antibody or fragmentthereof of the present disclosure comprises a nucleotide sequence thathas at least 80%, at least 85%, at least 90%, at least 95%, at least96%, 97%, 98%, or 99% sequence similarity to the nucleotide sequence ofSEQ ID NO: 3.

In one embodiment, the isolated polynucleotide of the disclosurecomprises the nucleotide sequences of SEQ ID NOs: 92, 98, and 104, ornucleotide sequences comprising at least 80%, at least 85%, at least90%, at least 95%, at least 96%, 97%, 98%, or 99% sequence similarity tothe nucleotide sequences of SEQ ID NOs: 92, 98, and 104. An exemplarypolynucleotide comprising the nucleotide sequences of SEQ ID NOs: 92,98, and 104 comprises the nucleotide sequence of SEQ ID NO: 7 (Table 5),which encodes the light chain variable region of the FT-12E1 antibody asdisclosed herein. In one embodiment, the isolated polynucleotideencoding the light chain variable region of the antibody or fragmentthereof of the present disclosure comprises a nucleotide sequence thathas at least 80%, at least 85%, at least 90%, at least 95%, at least96%, 97%, 98%, or 99% sequence similarity to the nucleotide sequence ofSEQ ID NO: 7.

In one embodiment, the isolated polynucleotide of the disclosurecomprises the nucleotide sequences of SEQ ID NOs: 93, 99, and 105, ornucleotide sequences comprising at least 80%, at least 85%, at least90%, at least 95%, at least 96%, 97%, 98%, or 99% sequence similarity tothe nucleotide sequences of SEQ ID NOs: 93, 99, and 105. An exemplarypolynucleotide comprising the nucleotide sequences of SEQ ID NOs: 93,99, and 105 comprises the nucleotide sequence of SEQ ID NO: 11 (Table5), which encodes the light chain variable region of the WG-3D7 antibodyas disclosed herein. In one embodiment, the isolated polynucleotideencoding the light chain variable region of the antibody or fragmentthereof of the present disclosure comprises a nucleotide sequence thathas at least 80%, at least 85%, at least 90%, at least 95%, at least96%, 97%, 98%, or 99% sequence similarity to the nucleotide sequence ofSEQ ID NO: 11.

In one embodiment, the isolated polynucleotide of the disclosurecomprises the nucleotide sequences of SEQ ID NOs: 94, 100, and 106, ornucleotide sequences comprising at least 80%, at least 85%, at least90%, at least 95%, at least 96%, 97%, 98%, or 99% sequence similarity tothe nucleotide sequences of SEQ ID NOs: 94, 100, and 106. An exemplarypolynucleotide comprising the nucleotide sequences of SEQ ID NOs: 94,100, and 106 comprises the nucleotide sequence of SEQ ID NO: 17 (Table5), which encodes the light chain variable region of the TF-10F7antibody as disclosed herein. In one embodiment, the isolatedpolynucleotide encoding the light chain variable region of the antibodyor fragment thereof of the present disclosure comprises a nucleotidesequence that has at least 80%, at least 85%, at least 90%, at least95%, at least 96%, 97%, 98%, or 99% sequence similarity to thenucleotide sequence of SEQ ID NO: 17.

In one embodiment, the isolated polynucleotide of the disclosurecomprises the nucleotide sequences of SEQ ID NOs: 95, 101, and 107, ornucleotide sequences comprising at least 80%, at least 85%, at least90%, at least 95%, at least 96%, 97%, 98%, or 99% sequence similarity tothe nucleotide sequences of SEQ ID NOs: 95, 101, and 107. An exemplarypolynucleotide comprising the nucleotide sequences of SEQ ID NOs: 95,101, and 107 comprises the nucleotide sequence of SEQ ID NO: 21 (Table5), which encodes the light chain variable region of the GW-23B7antibody as disclosed herein. In one embodiment, the isolatedpolynucleotide encoding the light chain variable region of the antibodyor fragment thereof of the present disclosure comprises a nucleotidesequence that has at least 80%, at least 85%, at least 90%, at least95%, at least 96%, 97%, 98%, or 99% sequence similarity to thenucleotide sequence of SEQ ID NO: 21.

In one embodiment, the isolated polynucleotide of the disclosurecomprises the nucleotide sequences of SEQ ID NOs: 96, 102, and 108, ornucleotide sequences comprising at least 80%, at least 85%, at least90%, at least 95%, at least 96%, 97%, 98%, or 99% sequence similarity tothe nucleotide sequences of SEQ ID NOs: 96, 102, and 108. An exemplarypolynucleotide comprising the nucleotide sequences of SEQ ID NOs: 96,102, and 108 comprises the nucleotide sequence of SEQ ID NO: 15 (Table5), which encodes the light chain variable region of the FT-11F2antibody (encoding the amino acid sequence of SEQ ID NO: 16) asdisclosed herein. In another embodiment, the isolated polynucleotide ofthe disclosure comprises the nucleotide sequence of SEQ ID NO: 110(Table 5), which encodes an alternative light chain variable region ofthe FT-11F2 (encoding the amino acid sequence of SEQ ID NO: 109). In oneembodiment, the isolated polynucleotide encoding the light chainvariable region of the antibody or fragment thereof of the presentdisclosure comprises a nucleotide sequence that has at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, 97%, 98%, or 99%sequence similarity to the nucleotide sequence of SEQ ID NO: 15 or SEQID NO: 110.

TABLE 5  Nucleotide Sequences Encoding V_(H) and V_(L) Antibody RegionsSEQ ID Antibody Region NO Sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4)TF-10E8 V_(H) 1 CAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACAGCTTCACAAGCTACTATATACACTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTGGAAGTGGTAATACTAAGTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACGGCAGACACATCCTCCAGCACTGCCTACATGCAGCTCAGCAGCCTAACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGGAGCTATGGTGACTACGACTACTGGGGCCAAGGCA CCACTCTCACAGTCTCCTCA TF-10E8V_(L) 3 GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCTCGGACGTTCGGTGGAGGCACCAAGCTGG AAATCAAA FT-12E1 V_(H) 5CAGGTGCAGCTGAAACAGTCAGGACCTGGCCTAGTGCCGCCCTCACAGAGCCTGTCCATCACCTGCACAGTTTCTGGTTTCTCATTAACTAGCTATGGTGTACACTGGGTTCGCCAGTCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTGATATGGAGTGGTGGAAGCACAGACTACAATGCAGCTTTCATATCCAGACTGAGCATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTTAAAATGAACAGTCTGCAAGCTGATGACACAGCCATATACTACTGTGCCAGAAATCCCTCCGCCTACTATAGTAACTACTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA FT-12E1 V_(L) 7GACATTGTGATGACCCAGTCTCAAAATTTCATGTCCACATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGTATGTGGGTACTTATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGGCATACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACCTGGCAGATTATTTCTGTCAGCAATATAGCAGCTCTCCTCTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA WG- V_(H) 9GAGGTCAAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTACT 3D7CATTCACTGGCTACTACATGCACTGGGTGAAGCAAAGTTCTGAAAAGAGCCTTGAGTGGATTGGAGAGATTAATCCTAGCACTGGTGGTACTAGCTACAACCAGAAGTTCAAGGGCAAGGCCACATTAACTGTAGACAAGTCATCCAGCACAGCCTACATGCAGCTCAAGAGCCTGACATCTGAGGACTCTGCTGTCTATTACTGTGCAAGAGACTACTATAGTAAGGCTTACTGGGGCCAAGGGA CTCTGGTCACTGTCTCTGCA WG-3D7V_(L) 11 AGTATTGTGATGACCCAGACTCCCAAATTCCTGCTTGTATCAGCAGGAGACAGGGTTACCATAACCTGCAAGGCCAGTCAGAGTGTGAGTAATGATGTAGCTTGGTACCAACAGAAGCCAGGGCAGTCTCCTAAACTGCTGATATACTATGCATCCAATCGCTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATATGGGACGGATTTCACTTTCACCATCAGCACTGTGCAGGCTGAAGACCTGGCAGTTTATTTCTGTCAGCAGGATTATAGCTCTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA TF-10F7 V_(H) 1CAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACAGCTTCACAAGCTACTATATACACTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTGGAAGTGGTAATACTAAGTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACGGCAGACACATCCTCCAGCACTGCCTACATGCAGCTCAGCAGCCTAACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGGAGCTATGGTGACTACGACTACTGGGGCCAAGGCA CCACTCTCACAGTCTCCTCA TF-10F7V_(L) 17 GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAA TCAAA GW-23B7 V_(H) 19GAGGTTCAGCTGCAGCAGTCTGTGGCAGAGCTTGTGAGGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAAACACCTATATGCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGTAATACTAAATATGCCCCGAAGTTCCAGGGCAAGGCCACTATAACTGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCATCTATTACTGTGCTAGAGGGAGTTTTTATGCTATGGACTACTGGGGTCAAG GAACCTCAGTCACCGTCTCCTCAGW-23B7 V_(L) 21 GATGTCCAGATAACCCAGTCTCCATCTTATCTTGCTGCATCTCCTGGAGAAACCATTACTATTAATTGCAGGGCAAGTAAGAGCATTAACAAATATTTAGCCTGGTATCAAGAGAAACCTGGGAAAACTAATAAGCTTCTTATCTACTCTGGATCCACCTTGCAATCTGGAATTCCATCAAGGTTCAGTGGCAGTGGATCTGGTACAGATTTTACTCTCACCATCAGTAGCCTGGAGCCTGAAGATTTTGCAATGTATCACTGTCAACAGCATAATGAATACCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA FT-11F2 V_(H) 13CAGGTCACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTATGGTATGGGTGTAGGTTGGATTCGTCAGCCTTCAGGGAAGGGTCTGGAGTGGCTGGCCAACATTTGGTGGAATGATGATAAGTACTATAACTCAGCCCTGAAGAGCCGGCTCACAATCTCCAAGGATACCTCCAACAACCAGGTATTCCTCAAGATCTCCAGTGTGGACACTGCAGATACTGCCACATACTACTGTGCTCAAATAGGGTGGTTACTAGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA FT-11F2 V_(L) 15GACATTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGTC (encodingAGCAGGAGAGAAGGTCACTATGAGCTGCAAATCCAGTCAGA the aminoGTCTGCTCAACAGTAGAACCCGAAAGAACTACTTGGCTTGG acidTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTA sequenceCTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCA of SEQ IDCAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGC NO: 16)AGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGCAAGCAATCTTATAATCTGCTCACGTTCGGTGCTGGGACCAAGCTGG AGCTGAAA FT-11F2 V_(L) 110GACATTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGTC (encodingAGCAGGAGACAAGGTCACTATGAGCTGCAAATCCAGTCAGA the aminoGTCTGCTCAACAGTAGAACCCGAAAGAACTACTTGGCTTGG acidTACCAGCAGAAACCAGGGCAGTCTCCTAAAGTGCTGGTCTA sequenceCTGGGGATCCACTAGGGACTCTGGGGTCCCTGATCGCTTCA of SEQ IDCAGGCAGTGGATCTGGGACAGATTACACTCTCACCGTCAGC NO: 109)AGTGTGCAGGCTGAAGACCTGGCAGTTTATTTCTGCAAGCAATCTTATAATCTGCTCACGTTCGGTGCTGGGACCAAGCTG

In another embodiment, exemplary polynucleotides include those encodinghumanized V_(H) and V_(L) regions as described supra. In anotherembodiment, exemplary polynucleotides include those encoding the heavychain and light chain components of the various antibodies describedherein, i.e., SEQ ID NOs: 56, 60, 64, 68, and 72, and SEQ ID NOs: 58,62, 66, 70, and 74, respectively, as disclosed in Table 6 below.

The polynucleotides of the invention may be produced by chemicalsynthesis such as solid phase polynucleotide synthesis on an automatedpolynucleotide synthesizer and assembled into complete single or doublestranded molecules. Alternatively, the polynucleotides of the inventionmay be produced by other techniques such a PCR followed by routinecloning. Techniques for producing or obtaining polynucleotides of agiven known sequence are well known in the art.

The polynucleotides of the invention may comprise at least onenon-coding sequence, such as a promoter or enhancer sequence, intron,polyadenylation signal, a cis sequence facilitating RepA binding, andthe like. The polynucleotide sequences may also comprise additionalsequences encoding additional amino acids that encode for example amarker or a tag sequence such as a histidine tag or an HA tag tofacilitate purification or detection of the protein, a signal sequence,a fusion protein partner such as RepA, Fc or bacteriophage coat proteinsuch as pIX or pIII.

Another embodiment of the disclosure is directed to a vector comprisingat least one polynucleotide as described herein. Such vectors may beplasmid vectors, viral vectors, vectors for baculovirus expression,transposon based vectors or any other vector suitable for introductionof the polynucleotides described herein into a given organism or geneticbackground by any means.

Another embodiment of the disclosure is directed to one or moreexpression vectors comprising the polynucleotides encoding the antibodyor binding fragment thereof or antibody mimetic as described herein. Thepolynucleotide sequences encoding the heavy and light chain variabledomains, Fab fragments, or full-length chains of the antibodiesdisclosed herein are combined with sequences of promoter, translationinitiation, 3′ untranslated region, polyadenylation, and transcriptiontermination to form one or more expression vector constructs.

In accordance with this embodiment, the expression vector constructencoding the antibodies or binding portions thereof as described hereincan include the nucleic acid encoding the heavy chain polypeptide, afragment thereof, a variant thereof, or combinations thereof. The heavychain polypeptide can include a variable heavy chain (VH) region and/orat least one constant heavy chain (CH) region. The at least one constantheavy chain region can include a constant heavy chain region 1 (CH1), aconstant heavy chain region 2 (CH2), a constant heavy chain region 3(CH3), a constant heavy chain region 4 (CH4) in the immunoglobulinclasses that it corresponds, and/or a hinge region. In some embodiments,the heavy chain polypeptide can include a VH region and a CH1 region. Inother embodiments, the heavy chain polypeptide can include a VH region,a CH1 region, a hinge region, a CH2 region, a CH3 region, and a CH4region.

The expression construct can also include a nucleic acid sequenceencoding the light chain polypeptide, a fragment thereof, a variantthereof, or combinations thereof. The light chain polypeptide caninclude a variable light chain (VL) region and/or a constant light chain(CL) region.

The expression construct also typically comprises a promoter sequencesuitable for driving expression of the antibody or binding fragmentthereof. Suitable promoter sequences include, without limitation, theelongation factor 1-alpha promoter (EF1a) promoter, a phosphoglyceratekinase-1 promoter (PGK) promoter, a cytomegalovirus immediate early genepromoter (CMV), a chimeric liver-specific promoter (LSP) acytomegalovirus enhancer/chicken beta-actin promoter (CAG), atetracycline responsive promoter (TRE), a transthyretin promoter (TTR),a simian virus 40 promoter (SV40) and a CK6 promoter. Other promoterssuitable for driving gene expression in mammalian cells that are knownin the art are also suitable for incorporation into the expressionconstructs disclosed herein.

The expression construct can further encode a linker sequence. Thelinker sequence can encode an amino acid sequence that spatiallyseparates and/or links the one or more components of the expressionconstruct (heavy chain and light chain components of the encodedantibody).

Another embodiment of the invention is a host cell comprising thevectors described herein. The antibodies and binding fragments thereofdescribed herein can be optionally produced by a cell line, a mixed cellline, an immortalized cell or clonal population of immortalized cells,as well known in the art (see e.g., Ausubel et al., ed., CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y.(1987-2001); Sambrook et al., Molecular Cloning: A Laboratory Manual,2^(nd) Edition, Cold Spring Harbor, N.Y. (1989); Harlow and Lane,Antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y. (1989);Colligan et al., eds., Current Protocols in Immunology, John Wiley &Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols inProtein Science, John Wiley & Sons, NY, N.Y., (1997-2001), which arehereby incorporated by reference in their entirety).

The host cell chosen for expression may be of mammalian origin or may beselected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, He G2, SP2/0,HeLa, myeloma, lymphoma, yeast, insect, or plant cells, or anyderivative, immortalized or transformed cell thereof. Alternatively, thehost cell may be selected from a species or organism incapable ofglycosylating polypeptides, e.g., a prokaryotic cell or organism, suchas BL21, BL21(DE3), BL21-GOLD(DE3), XL1-Blue, JM109, HMS174,HMS174(DE3), and any of the natural or engineered E. coli spp,Klebsiella spp., or Pseudomonas spp strains.

The antibodies described herein can be prepared by any of a variety oftechniques using the isolated polynucleotides, vectors, and host cellsdescribed supra. In general, antibodies can be produced by cell culturetechniques, including the generation of monoclonal antibodies viaconventional techniques, or via transfection of antibody genes, heavychains and/or light chains into suitable bacterial or mammalian cellhosts, in order to allow for the production of antibodies, wherein theantibodies may be recombinant. Standard molecular biology techniques areused to prepare the recombinant expression vector, transfect the hostcells, select for transformants, culture the host cells and recover theantibody from the culture medium. Transfecting the host cell can becarried out using a variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., by electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is possible toexpress the antibodies described herein in either prokaryotic oreukaryotic host cells, expression of antibodies in eukaryotic cells issometimes preferable, and sometimes preferable in mammalian host cells,because such eukaryotic cells (and in particular mammalian cells) aremore likely than prokaryotic cells to assemble and secrete a properlyfolded and immunologically active antibody.

As noted above, exemplary mammalian host cells for expressing therecombinant antibodies of the invention include Chinese Hamster Ovary(CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin,Proc. Natl. Acad. Sci. USA, 77: 4216-4220 (1980), which is herebyincorporated by reference in its entirety). Other suitable mammalianhost cells include, without limitation, NS0 myeloma cells, COS cells,and SP2 cells. When recombinant expression vectors encoding antibodygenes are introduced into mammalian host cells, the antibodies areproduced by culturing the host cells for a period of time sufficient toallow for expression of the antibody in the host cells or, morepreferably, secretion of the antibody into the culture medium in whichthe host cells are grown.

Host cells can also be used to produce functional antibody fragments,such as Fab fragments or scFv molecules. It is understood thatvariations on the above procedure are within the scope of the presentdisclosure. For example, it may be desirable to transfect a host cellwith DNA encoding functional fragments of either the light chain and/orthe heavy chain of an antibody described herein. Recombinant DNAtechnology may also be used to remove some or all the DNA encodingeither or both of the light and heavy chains that is not necessary forbinding to the antigens of interest. The molecules expressed from suchtruncated DNA molecules are also encompassed by the antibodies describedherein.

In one embodiment, the sequence of the polynucleotide molecules encodingthe antibodies and binding fragments described herein are modified usinggene editing technology. Suitable gene editing technology and systemsinclude, for example, zinc finger nucleases (“ZFNs”) (Urnov et at,“Genome Editing with Engineered Zinc Finger Nucleases,” Nat. Rev. Genet.11: 636-646 (2010), which is hereby incorporated by reference in itsentirety), transcription activator-like effector nucleases (“TALENs”)(Joung & Sander, “TALENs: A Widely Applicable Technology for TargetedGenome Editing,” Nat. Rev. Mot. Cell Biol. 14: 49-55 (2013), which ishereby incorporated by reference in its entirety), clustered regularlyinterspaced short palindromic repeat (“CRISPR”)-associated endonucleases(e.g., CRISPR/CRISPR-associated (“Cas”) 9 systems) (Wiedenheft et al.,“RNA-Guided Genetic Silencing Systems in Bacteria and Archaea,” Nature482:331-338 (2012); Zhang et al., “Multiplex Genome Engineering UsingCRISPR/Cas Systems,” Science 339(6121): 819-23 (2013); and Gaj et al.,“ZFN, TALEN, and CRISPR/Cas-based Methods for Genome Engineering,” Cell31(7):397-405 (2013), which are hereby incorporated by reference intheir entirety). Gene editing modifications can be employed forhumanization, class-switch recombination, and/or antibody fragmentproduction (see e.g., Cheong et al., “Editing of Mouse and HumanImmunoglobulin Genes by CRISPR-Cas9 System,” Nature Comm. 7: 10934(2016), and Flisikowska et al., “Efficient Immunoglobulin GeneDisruption and Targeted Replacement in Rabbit Using Zinc FingerNucleases,” PLOS One 6(6): e21045 (2011), which are hereby incorporatedby reference in their entirety.

The antibodies and antibody binding fragments are recovered and purifiedfrom recombinant cell cultures by known methods including, but notlimited to, protein A purification, ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. High performance liquid chromatography (“HPLC”) can alsobe used for purification.

Table 6 below provides the heavy chain and light chain amino acid andcorresponding nucleotide sequences of the exemplary antibodies that aredescribed herein.

TABLE 6  Heavy Chain (HC) and Light Chain (LC) Nucleotide and Amino AcidSequences of Exemplary Antibodies of the Disclosure SEQ SEQUENCEAntibody/ ID Leader sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4- DescriptionNO Constant region-Stop codon TF-10E8 56ATGGGATGGAGCTGGATCTTTCTCTTCCTCCTGTCAGGAACTGC HC DNAAGGTGTCCATTGCCAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACAGCTTCACAAGCTACTATATACAC TGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGA TGGATTTATCCTGGAAGTGGTAATACTAAGTACAATGAGAAGTTCAAGGGC AAGGCCACACTGACGGCAGACACATCCTCCAGCACTGCCTACATGCAGCTCAGCAGCCTAACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGGA GCTATGGTGACTACGACTACTGGGGCCAAGGCACCACTCTCACA GTCTCCTCA GAGAGTCAGTCCTTCCCAAATGTCTTTCCCCTCGTCTCCTGCGAGAGCCCCCTGTCTGATAAGAATCTGGTGGCCATGGGCTGCCTGGCCCGGGACTTCCTGCCCAGCACCATTTCCTTCACCTGGAACTACCAGAACAACACTGAAGTCATCCAGGGTATCAGAACCTTCCCAACACTGAGGACAGGGGGCAAGTACCTAGCCACCTCGCAGGTGTTGCTGTCTCCCAAGAGCATCCTTGAAGGTTCAGATGAATACCTGGTATGCAAAATCCACTACGGAGGCAAAAACAGAGATCTGCATGTGCCCATTCCAGCTGTCGCAGAGATGAACCCCAATGTAAATGTGTTCGTCCCACCACGGGATGGCTTCTCTGGCCCTGCACCACGCAAGTCTAAACTCATCTGCGAGGCCACGAACTTCACTCCAAAACCGATCACAGTATCCTGGCTAAAGGATGGGAAGCTCGTGGAATCTGGCTTCACCACAGATCCGGTGACCATCGAGAACAAAGGATCCACACCCCAAACCTACAAGGTCATAAGCACACTTACCATCTCTGAAATCGACTGGCTGAACCTGAATGTGTACACCTGCCGTGTGGATCACAGGGGTCTCACCTTCTTGAAGAACGTGTCCTCCACATGTGCTGCCAGTCCCTCCACAGACATCCTAACCTTCACCATCCCCCCCTCCTTTGCCGACATCTTCCTCAGCAAGTCCGCTAACCTGACCTGTCTGGTCTCAAACCTGGCAACCTATGAAACCCTGAATATCTCCTGGGCTTCTCAAAGTGGTGAACCACTGGAAACCAAAATTAAAATCATGGAAAGTCATCCCAATGGCACCTTCAGTGCTAAGGGTGTGGCTAGTGTTTGTGTGGAAGACTGGAATAACAGGAAGGAATTTGTGTGTACTGTGACTCACAGGGATCTGCCTTCACCACAGAAGAAATTCATCTCAAAACCCAATGAGGTGCACAAACATCCACCTGCTGTGTACCTGCTGCCACCAGCTCGTGAGCAACTGAACCTGAGGGAGTCAGCCACAGTCACCTGCCTGGTGAAGGGCTTCTCTCCTGCAGACATCAGTGTGCAGTGGCTTCAGAGAGGGCAACTCTTGCCCCAAGAGAAGTATGTGACCAGTGCCCCGATGCCAGAGCCTGGGGCCCCAGGCTTCTACTTTACCCACAGCATCCTGACTGTGACAGAGGAGGAATGGAACTCCGGAGAGACCTATACCTGTGTTGTAGGCCACGAGGCCCTGCCACACCTGGTGACCGAGAGGACCGTGGACAAGTCCACTGGTAAACCCACACTGTACAATGTCTCCCTGATCATGTCTGACACAGGCGG CACCTGCTAT TGA TF-10E8 57MGWSWIFLFLLSGTAGVHCQVQLQQSGPELVKPGASVKISCKAS HC GYSFTSYYIHWVKQRPGQGLEWIG WIYPGSGNTKYNEKFKG KAT Amino AcidLTADTSSSTAYMQLSSLTSEDSAVYYCAR SYGDYDY WGQGTTLT VSSESQSFPNVFPLVSCESPLSDKNLVAMGCLARDFLPSTISFTWNYQNNTEVIQGIRTFPTLRTGGKYLATSQVLLSPKSILEGSDEYLVCKIHYGGKNRDLHVPIPAVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKEVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNENVYTCRVDERGLTFLKNVSSTCAASPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKTKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPNEVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADTSVQWLQRGQLLPQEKYVISAPMPEPGAPGFYETHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTGK PTLYNVSLIMSDTGGTCYTF-10E8 LC  58 ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCC DNATGCTTCCAGCAGTGATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGC AGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACAT TGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTAC A AAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGC AGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGC TCTCAAAGTACACATG TTCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAG GAATGAGTGT TAG TF-10E8 LC59 MKLPVRLLVLMFWIPASSSDVVMTQTPLSLPVSLGDQASISC RS Amino AcidSQSLVHSNGNTYLH WYLQKPGQSPKLLIY KVSNRFS GVPDRFSG SGSGTDFTLKISRVEAEDLGVYFCSQSTHVPRT FGGGTKLEIK R ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEA THKTSTSPIVKSFNRNEC FT-12E160 ATGGCTGTCCTGGTGCTGCTCCTCTGCCTGGTGACATTCCCAAG HC DNACTGTGTCCTGTCCCAGGTGCAGCTGAAACAGTCAGGACCTGGCCTAGTGCCGCCCTCACAGAGCCTGTCCATCACCTGCACAGTTTCTGGTTTCTCATTAACTAGCTATGGTGTACAC TGGGTTCGCCAGTCTCCAGGAAAGGGTCTGGAGTGGCTGGGA GTGATATGGAGTGGTGGAAGCACAGACTACAATGCAGCTTTCATATCC AGACTGAGCATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTTAAAATGAACAGTCTGCAAGCTGATGACACAGCCATATACTACTGTGCCAGAAATCCCTCCGCCTACTATAGTAACTACTGGTTTGCTAAC TGGGGCCAA GGGACTCTGGTCACTGTCTCTGCAGAGAGTCAGTCCTTCCCAAA TGTCTTTCCCCTCGTCTCCTGCGAGAGCCCCCTGTCTGATAAGAATCTGGTGGCCATGGGCTGCCTGGCCCGGGACTTCCTGCCCAGCACCATTTCCTTCACCTGGAACTACCAGAACAACACTGAAGTCATCCAGGGTATCAGAACCTTCCCAACACTGAGGACAGGGGGCAAGTACCTAGCCACCTCGCAGGTGTTGCTGTCTCCCAAGAGCATCCTTGAAGGTTCAGATGAATACCTGGTATGCAAAATCCACTACGGAGGCAAAAACAGAGATCTGCATGTGCCCATTCCAGCTGTCGCAGAGATGAACCCCAATGTAAATGTGTTCGTCCCACCACGGGATGGCTTCTCTGGCCCTGCACCACGCAAGTCTAAACTCATCTGCGAGGCCACGAACTTCACTCCAAAACCGATCACAGTATCCTGGCTAAAGGATGGGAAGCTCGTGGAATCTGGCTTCACCACAGATCCGGTGACCATCGAGAACAAAGGATCCACACCCCAAACCTACAAGGTCATAAGCACACTTACCATCTCTGAAATCGACTGGCTGAACCTGAATGTGTACACCTGCCGTGTGGATCACAGGGGTCTCACCTTCTTGAAGAACGTGTCCTCCACATGTGCTGCCAGTCCCTCCACAGACATCCTAACCTTCACCATCCCCCCCTCCTTTGCCGACATCTTCCTCAGCAAGTCCGCTAACCTGACCTGTCTGGTCTCAAACCTGGCAACCTATGAAACCCTGAATATCTCCTGGGCTTCTCAAAGTGGTGAACCACTGGAAACCAAAATTAAAATCATGGAAAGTCATCCCAATGGCACCTTCAGTGCTAAGGGTGTGGCTAGTGTTTGTGTGGAAGACTGGAATAACAGGAAGGAATTTGTGTGTACTGTGACTCACAGGGATCTGCCTTCACCACAGAAGAAATTCATCTCAAAACCCAATGAGGTGCACAAACATCCACCTGCTGTGTACCTGCTGCCACCAGCTCGTGAGCAACTGAACCTGAGGGAGTCAGCCACAGTCACCTGCCTGGTGAAGGGCTTCTCTCCTGCAGACATCAGTGTGCAGTGGCTTCAGAGAGGGCAACTCTTGCCCCAAGAGAAGTATGTGACCAGTGCCCCGATGCCAGAGCCTGGGGCCCCAGGCTTCTACTTTACCCACAGCATCCTGACTGTGACAGAGGAGGAATGGAACTCCGGAGAGACCTATACCTGTGTTGTAGGCCACGAGGCCCTGCCACACCTGGTGACCGAGAGGACCGTGGACAAGTCCACTGGTAAACCCACACTGTACAATGTCTCCCTGATCAT GTCTGACACAGGCGGCACCTGCTATTGA FT-12E1 61 MAVLVLLLCLVTFPSCVLSQVQLKQSGPGLVPPSQSLSITCTVS HC aminoGFSLTSYGVH WVRQSPGKGLEWLG VIWSGGSTDYNAAFIS RLSI acidSKDNSKSQVFFKMNSLQADDTAIYYCARNPSAYYSNYWFAYWGQ GTLVTVSAESQSFPNVFPLVSCESPLSDKNLVAMGCLARDFLPSTISFTWNYQNNTEVIQGIRTFPTLRTGGKYLATSQVLLSPKSILEGSDEYLVCKIHYGGKNRDLHVPIPAVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCAASPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPNEVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVD KSTGKPTLYNVSLIMSDTGGTCYFT-12E1  62 ATGGAGACACATTCTCAGGTCTTTGTATACATGTTGCTGTGGTT LC DNAGTCTGGTGTTGATGGAGACATTGTGATGACCCAGTCTCAAAATTTCATGTCCACATCAGTAGGAGACAGGGTCAGCGTCACCTGC AAGGCCAGTCAGTATGTGGGTACTTATGTAGCC TGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTAC TCGGCATCCTACCGGCATACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACCT GGCAGATTATTTCTGTCAGCAATATAGCAGCTCTCCTCTCACG T TCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCA CCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAGAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGTGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACATTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATCGTCAAGAGCTTCAACAGGAATGAGTGT TA G FT-12E1 LC 63METHSQVFVYMLLWLSGVDGDIVMTQSQNFMSTSVGDRVSVTC K amino acid ASQYVGTYVAWYQQKPGQSPKALIY SASYRHT GVPDRFTGSGSG TDFTLTISNVQSEDLADYFC QQYSSSPLTFGSGTKLEIK RADAA PTVSIFSPSSEQLTSGGASVVCFLNNFYPRDINVKWKTDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKT STSPIVKSFNRNEC WG-3D7 64ATGGAATGGAGCTGGGTCTTTCTCTTCCTCCTGTCAGTAACTAC HC DNAAGGTGTCCACTCTGAGGTCAAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACTACATGCAC TGGGTGAAGCAAAGTTCTGAAAAGAGCCTTGAGTGGATTGGA GAGATTAATCCTAGCACTGGTGGTACTAGCTACAACCAGAAGTTCAAGGGC AAGGCCACATTAACTGTAGACAAGTCATCCAGCACAGCCTACATGCAGCTCAAGAGCCTGACATCTGAGGACTCTGCTGTCTATTACTGTGCAAGAG ACTACTATAGTAAGGCTTACTGGGGCCAAGGGACTCTGGTCACT GTCTCTGCA GAGAGTCAGTCCTTCCCAAATGTCTTTCCCCTCGTCTCCTGCGAGAGCCCCCTGTCTGATAAGAATCTGGTGGCCATGGGCTGCCTGGCCCGGGACTTCCTGCCCAGCACCATTTCCTTCACCTGGAACTACCAGAACAACACTGAAGTCATCCAGGGTATCAGAACCTTCCCAACACTGAGGACAGGGGGCAAGTACCTAGCCACCTCGCAGGTGTTGCTGTCTCCCAAGAGCATCCTTGAAGGTTCAGATGAATACCTGGTATGCAAAATCCACTACGGAGGCAAAAACAGAGATCTGCATGTGCCCATTCCAGCTGTCGCAGAGATGAACCCCAATGTAAATGTGTTCGTCCCACCACGGGATGGCTTCTCTGGCCCTGCACCACGCAAGTCTAAACTCATCTGCGAGGCCACGAACTTCACTCCAAAACCGATCACAGTATCCTGGCTAAAGGATGGGAAGCTCGTGGAATCTGGCTTCACCACAGATCCGGTGACCATCGAGAACAAAGGATCCACACCCCAAACCTACAAGGTCATAAGCACACTTACCATCTCTGAAATCGACTGGCTGAACCTGAATGTGTACACCTGCCGTGTGGATCACAGGGGTCTCACCTTCTTGAAGAACGTGTCCTCCACATGTGCTGCCAGTCCCTCCACAGACATCCTAACCTTCACCATCCCCCCCTCCTTTGCCGACATCTTCCTCAGCAAGTCCGCTAACCTGACCTGTCTGGTCTCAAACCTGGCAACCTATGAAACCCTGAATATCTCCTGGGCTTCTCAAAGTGGTGAACCACTGGAAACCAAAATTAAAATCATGGAAAGTCATCCCAATGGCACCTTCAGTGCTAAGGGTGTGGCTAGTGTTTGTGTGGAAGACTGGAATAACAGGAAGGAATTTGTGTGTACTGTGACTCACAGGGATCTGCCTTCACCACAGAAGAAATTCATCTCAAAACCCAATGAGGTGCACAAACATCCACCTGCTGTGTACCTGCTGCCACCAGCTCGTGAGCAACTGAACCTGAGGGAGTCAGCCACAGTCACCTGCCTGGTGAAGGGCTTCTCTCCTGCAGACATCAGTGTGCAGTGGCTTCAGAGAGGGCAACTCTTGCCCCAAGAGAAGTATGTGACCAGTGCCCCGATGCCAGAGCCTGGGGCCCCAGGCTTCTACTTTACCCACAGCATCCTGACTGTGACAGAGGAGGAATGGAACTCCGGAGAGACCTATACCTGTGTTGTAGGCCACGAGGCCCTGCCACACCTGGTGACCGAGAGGACCGTGGACAAGTCCACTGGTAAACCCACACTGTACAATGTCTCCCTGATCATGTCTGACACAGGCGG CACCTGCTAT TGA WG-3D7 65MEWSWVFLFLLSVTTGVHSEVKLQQSGPELVKPGASVKISCKAS HC amino GYSFTGYYMHWVKQSSEKSLEWIG EINPSTGGTSYNQKFKG KAT acid LTVDKSSSTAYMQLKSLTSEDSAVYYCARDYYSKAY WGQGTLVT VSA ESQSFPNVFPLVSCESPLSDKNLVAMGCLARDFLPSTISFTWNYQNNTEVIQGIRTFPTLRTGGKYLATSQVLLSRKSILEGSDEYLVCKTHYGGKNRDLHVPIPAVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNINVYTCRVDHRGLTFLKNVSSTCAASPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPOKKEISKPNEVEKHPPAVYLLPPAREOLNLRESATVTCLVKGESPADISVQWLQRGQLLPQEKYVISAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTGK PTLYNVSLIMSDTGGTCY WG-3D766 ATGAAGTCACAGACCCAGGTCTTCGTATTTCTACTGCTCTGTGT LC DNAGTCTGGTGCTCATGGGAGTATTGTGATGACCCAGACTCCCAAATTCCTGCTTGTATCAGCAGGAGACAGGGTTACCATAACCTGC AAGGCCAGTCAGAGTGTGAGTAATGATGTAGCT TGGTACCAACAGAAGCCAGGGCAGTCTCCTAAACTGCTGATATAC TATGCATCCAATCGCTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATATGGGACGGATTTCACTTTCACCATCAGCACTGTGCAGGCTGAAGACCT GGCAGTTTATTTCTGTCAGCAGGATTATAGCTCTCCGTACACG T TCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCA CCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT TA G WG-3D7 67MKSQTQVFVFLLLCVSGAHGSIVMTQTPKFLLVSAGDRVTITC K LC amino ASQSVSNDVAWYQQKPGQSPKLLIY YASNRYT GVPDRFTGSGYG acid TDFTFTISTVQAEDLAVYFC QQDYSSPYTFGGGTKLEIK RADAA PTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKT STSPIVKSFNRNEC TF-10F7 68ATGGGATGGAGCTGGATCTTTCTCTTCCTCCTGTCAGGAACTGC HC DNAAGGTGTCCATTGCCAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACAGCTTCACAAGCTACTATATACAC TGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGA TGGATTTATCCTGGAAGTGGTAATACTAAGTACAATGAGAAGTTCAAGGGC AAGGCCACACTGACGGCAGACACATCCTCCAGCACTGCCTACATGCAGCTCAGCAGCCTAACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGGA GCTATGGTGACTACGACTACTGGGGCCAAGGCACCACTCTCACA GTCTCCTCA GAGAGTCAGTCCTTCCCAAATGTCTTTCCCCTCGTCTCCTGCGAGAGCCCCCTGTCTGATAAGAATCTGGTGGCCATGGGCTGCCTGGCCCGGGACTTCCTGCCCAGCACCATTTCCTTCACCTGGAACTACCAGAACAACACTGAAGTCATCCAGGGTATCAGAACCTTCCCAACACTGAGGACAGGGGGCAAGTACCTAGCCACCTCGCAGGTGTTGCTGTCTCCCAAGAGCATCCTTGAAGGTTCAGATGAATACCTGGTATGCAAAATCCACTACGGAGGCAAAAACAGAGATCTGCATGTGCCCATTCCAGCTGTCGCAGAGATGAACCCCAATGTAAATGTGTTCGTCCCACCACGGGATGGCTTCTCTGGCCCTGCACCACGCAAGTCTAAACTCATCTGCGAGGCCACGAACTTCACTCCAAAACCGATCACAGTATCCTGGCTAAAGGATGGGAAGCTCGTGGAATCTGGCTTCACCACAGATCCGGTGACCATCGAGAACAAAGGATCCACACCCCAAACCTACAAGGTCATAAGCACACTTACCATCTCTGAAATCGACTGGCTGAACCTGAATGTGTACACCTGCCGTGTGGATCACAGGGGTCTCACCTTCTTGAAGAACGTGTCCTCCACATGTGCTGCCAGTCCCTCCACAGACATCCTAACCTTCACCATCCCCCCCTCCTTTGCCGACATCTTCCTCAGCAAGTCCGCTAACCTGACCTGTCTGGTCTCAAACCTGGCAACCTATGAAACCCTGAATATCTCCTGGGCTTCTCAAAGTGGTGAACCACTGGAAACCAAAATTAAAATCATGGAAAGTCATCCCAATGGCACCTTCAGTGCTAAGGGTGTGGCTAGTGTTTGTGTGGAAGACTGGAATAACAGGAAGGAATTTGTGTGTACTGTGACTCACAGGGATCTGCCTTCACCACAGAAGAAATTCATCTCAAAACCCAATGAGGTGCACAAACATCCACCTGCTGTGTACCTGCTGCCACCAGCTCGTGAGCAACTGAACCTGAGGGAGTCAGCCACAGTCACCTGCCTGGTGAAGGGCTTCTCTCCTGCAGACATCAGTGTGCAGTGGCTTCAGAGAGGGCAACTCTTGCCCCAAGAGAAGTATGTGACCAGTGCCCCGATGCCAGAGCCTGGGGCCCCAGGCTTCTACTTTACCCACAGCATCCTGACTGTGACAGAGGAGGAATGGAACTCCGGAGAGACCTATACCTGTGTTGTAGGCCACGAGGCCCTGCCACACCTGGTGACCGAGAGGACCGTGGACAAGTCCACTGGTAAACCCACACTGTACAATGTCTCCCTGATCATGTCTGACACAGG CGGCACCTGCTAT TGA TF-10F769 MGWSWIFLFLLSGTAGVHCQVQLQQSGPELVKPGASVKISCKAS HC amino GYSFTSYYIHWVKQRPGQGLEWIG WIYPGSGNTKYNEKFKG KAT acid LTADTSSSTAYMQLSSLTSEDSAVYYCARSYGDYDY WGQGTTLT VSS ESQSFPNVFPLVSCESPLSDKNLVAMGCLARDFLPSTISFTWNYQNNTEVIQGIRTFPTLRTGGKYLATSQVLLSPKSTLEGSDEYLVCKIHYGGKNRDLHVPIPAVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNENVYTCRVDHRGLTFLKNVSSTCAASPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPNEVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYETHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTGK PTLYNVSLIMSDTGGTCY TF-10F770 ATGGAGACAGACACACTCCTGTTATGGGTACTGCTGCTCTGGGT LC DNATCCAGGTTCCACTGGTGACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATAC AGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCAC TGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTAT C TTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGC AGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGC TCTCAAAGTACACATG TTCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAG GAATGAGTGTTAG TF-10F7 71METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISY R LC amino ASKSVSTSGYSYMHWNQQKPGQPPRLLIY LVSNLES GVPARFSG acid SGSGTDFTLKISRVEAEDLGVYFC SQSTHVPRTFGGGTKLEIK R ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERONGVINSWIDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEA THKTSTSPIVKSFNRNEC GW-23B772 ATGAAATTCAGCTGGGTCATCTTCTTCCTGATGGCAGTGGTTAC HC DNAAGGGGTCAATTCAGAGGTTCAGCTGCAGCAGTCTGTGGCAGAGCTTGTGAGGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAAACACCTATATGCAC TGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGA AGGATTGATCCTGCGAATGGTAATACTAAATATGCCCCGAAGTTCCAGGGC AAGGCCACTATAACTGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCATCTATTACTGTGCTAGAG GGAGT TTTTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTC ACCGTCTCCTCA GAGAGTCAGTCCTTCCCAAATGTCTTTCCCCTCGTCTCCTGCGAGAGCCCCCTGTCTGATAAGAATCTGGTGGCCATGGGCTGCCTGGCCCGGGACTTCCTGCCCAGCACCATTTCCTTCACCTGGAACTACCAGAACAACACTGAAGTCATCCAGGGTATCAGAACCTTCCCAACACTGAGGACAGGGGGCAAGTACCTAGCCACCTCGCAGGTGTTGCTGTCTCCCAAGAGCATCCTTGAAGGTTCAGATGAATACCTGGTATGCAAAATCCACTACGGAGGCAAAAACAGAGATCTGCATGTGCCCATTCCAGCTGTCGCAGAGATGAACCCCAATGTAAATGTGTTCGTCCCACCACGGGATGGCTTCTCTGGCCCTGCACCACGCAAGTCTAAACTCATCTGCGAGGCCACGAACTTCACTCCAAAACCGATCACAGTATCCTGGCTAAAGGATGGGAAGCTCGTGGAATCTGGCTTCACCACAGATCCGGTGACCATCGAGAACAAAGGATCCACACCCCAAACCTACAAGGTCATAAGCACACTTACCATCTCTGAAATCGACTGGCTGAACCTGAATGTGTACACCTGCCGTGTGGATCACAGGGGTCTCACCTTCTTGAAGAACGTGTCCTCCACATGTGCTGCCAGTCCCTCCACAGACATCCTAACCTTCACCATCCCCCCCTCCTTTGCCGACATCTTCCTCAGCAAGTCCGCTAACCTGACCTGTCTGGTCTCAAACCTGGCAACCTATGAAACCCTGAATATCTCCTGGGCTTCTCAAAGTGGTGAACCACTGGAAACCAAAATTAAAATCATGGAAAGTCATCCCAATGGCACCTTCAGTGCTAAGGGTGTGGCTAGTGTTTGTGTGGAAGACTGGAATAACAGGAAGGAATTTGTGTGTACTGTGACTCACAGGGATCTGCCTTCACCACAGAAGAAATTCATCTCAAAACCCAATGAGGTGCACAAACATCCACCTGCTGTGTACCTGCTGCCACCAGCTCGTGAGCAACTGAACCTGAGGGAGTCAGCCACAGTCACCTGCCTGGTGAAGGGCTTCTCTCCTGCAGACATCAGTGTGCAGTGGCTTCAGAGAGGGCAACTCTTGCCCCAAGAGAAGTATGTGACCAGTGCCCCGATGCCAGAGCCTGGGGCCCCAGGCTTCTACTTTACCCACAGCATCCTGACTGTGACAGAGGAGGAATGGAACTCCGGAGAGACCTATACCTGTGTTGTAGGCCACGAGGCCCTGCCACACCTGGTGACCGAGAGGACCGTGGACAAGTCCACTGGTAAACCCACACTGTACAATGTCTCCCTGATCATGTCTGACACAGG CGGCACCTGCTAT TGA GW-23B773 MKFSWVIFFLMAVVTGVNSEVQLQQSVAELVRPGASVKLSCTAS HC amino GFNIKNTYMHWVKQRPEQGLEWIG RIDPANGNTKYAPKFQG KAT acidITADTSSNTAYLQLSSLTSEDTAIYYCARGS FYAMDY WGQGTSV TVSSESQSFPNVFPLVSCESPLSDKNLVAMGCLARDFLPSTISFTWNYQNNTEVIOGIRTEPTLRTGGKYLATSQVLLSPKSILEGSDEYLVCKIHYGGKNRDLHVPIPAVAEMNPNVNVFVPPRDGFSGPAPRKSKTICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCAASPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPNEVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTG KPTLYNVSLIMSDTGGTCY GW-23B774 ATGAGGTTCCAGGTTCAGGTTCTGGGGCTCCTTCTGCTCTGGAT LC DNAACCAGGTGCCCAGTGTGATGTCCAGATAACCCAGTCTCCATCTTATCTTGCTGCATCTCCTGGAGAAACCATTACTATTAATTGC AGGGCAAGTAAGAGCATTAACAAATATTTAGCC TGGTATCAAGAGAAACCTGGGAAAACTAATAAGCTTCTTATCTAC TCTGGATCCACCT TGCAATCTGGAATTCCATCAAGGTTCAGTGGCAGTGGATCTGGTACAGATTTTACTCTCACCATCAGTAGCCTGGAGCCTGAAGATTT TGCAATGTATCACTGTCAACAGCATAATGAATACCCGTGGACG T TCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCA CCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAGAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGTGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACATTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATCGTCAAGAGCTTCAACAGGAATGAGTGT TA G GW-23B7 75MRFQVQVLGLLLLWIPGAQCDVQITQSPSYLAASPGETITINCR LC amino ASKSINKYLAWYQEKPGKTNKLLIY SGSTLQS GIPSRFSGSGSG acid TDFTLTISSLEPEDFAMYHC QQHNEYPWTFGGGTKLEIK RADAA PTVSIFSPSSEQLTSGGASVVCFLNNFYPRDINVKWKTDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKT STSPIVKSFNRNEC

In another embodiment, the antibody or binding fragment thereof is acomponent of a pharmaceutical composition. In one embodiment, thepharmaceutical composition comprises a monoclonal antibody composition.In another embodiment, the pharmaceutical composition comprises one ormore different antibodies as described herein. In another embodiment,the pharmaceutical composition comprises at least two differentantibodies that bind to different epitopes of toxic oligomeric forms ofamyloidogenic proteins or peptides as described herein. In anotherembodiment, the pharmaceutical composition comprises one or moreantibodies as described herein and one or more prophylactic ortherapeutic agents other than the antibodies described herein that areuseful for preventing or treating a condition mediated by anamyloidogenic protein or peptide.

The therapeutically effective amount of antibody present in thepharmaceutical composition or formulation is determined by taking intoaccount the desired dose volumes and mode(s) of administration.Exemplary antibody concentrations in the pharmaceutical compositions ofthe present disclosure include from about 0.1 mg/mL to about 50 mg/mL,from about 0.5 mg/mL to about 25 mg/mL, and from about 2 mg/mL to about10 mg/mL.

An aqueous formulation is prepared comprising the antibody in apH-buffered solution. The buffer has a pH in the range from about 4.5 toabout 10, from about 5 to about 9, or from about 6 to about 8. Examplesof buffers include phosphate buffers (e.g., phosphate buffered saline),acetate (e.g. sodium acetate), succinate (such as sodium succinate),gluconate, histidine, citrate and other organic acid buffers.

A polyol, which acts as a tonicifier and may stabilize the antibody, maybe included in the formulation. In one embodiment, the tonicifyingpolyol is a salt such as sodium chloride. In another embodiment, thepolyol is a nonreducing sugar, such as sucrose or trehalose. The polyolis added to the formulation in an amount which may vary with respect tothe desired isotonicity of the formulation. Preferably the aqueousformulation is isotonic, in which case suitable concentrations of thepolyol in the formulation are in the range from about 1% to about 15%w/v, or in the range from about 2% to about 10% w/v, for example.However, hypertonic or hypotonic formulations may also be suitable. Theamount of polyol added may also alter with respect to the molecularweight of the polyol. For example, a lower amount of a monosaccharide(e.g. mannitol) may be added, compared to a disaccharide (such astrehalose).

A surfactant may also be added to the pharmaceutical compositioncontaining the antibody. Exemplary surfactants include nonionicsurfactants such as polysorbates (e.g. polysorbates 20, 80 etc),poloxamers (e.g. poloxamer 188), Pluronic F68, and PEG (polyethyleneglycol). The amount of surfactant added is such that it reducesaggregation of the formulated antibody and/or minimizes the formation ofparticulates in the formulation and/or reduces adsorption. For example,the surfactant may be present in the formulation in an amount from about0.001% to about 0.5%, from about 0.005% to about 0.2%, or from about0.01% to about 0.1%.

In one embodiment, the pharmaceutical composition contains theabove-identified agents (i.e. antibody, buffer, polyol and surfactant)and is essentially free of one or more preservatives, such as benzylalcohol, phenol, m-cresol, chlorobutanol and benzethonium Cl. In anotherembodiment, a preservative may be included in the pharmaceuticalcomposition, particularly where the formulation is a multidoseformulation. Suitable preservatives include, without limitation phenol,m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol,phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol,magnesium chloride (e.g., hexahydrate), alkylparaben (methyl, ethyl,propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal, or mixtures thereof inan aqueous diluent. The concentration of preservative may be in therange from about 0.01% to about 5%, from about 0.5% to about 2% and anyrange or value therein. Non-limiting examples include, no preservative,0.1-2% m-cresol, 0.1-3% benzyl alcohol, 0.001-0.5% thimerosal,0.001-2.0% phenol, 0.0005-1.0% alkylparaben(s), and the like. One ormore other pharmaceutically acceptable carriers, excipients orstabilizers such as those described in Remington's PharmaceuticalSciences 16th edition, Osol, A. Ed. (1980) may be included in thecomposition provided that they do not adversely affect the desiredcharacteristics of the formulation. Acceptable carriers, excipients orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed and include; additional buffering agents; co-solvents;antioxidants including ascorbic acid and methionine; chelating agentssuch as EDTA; metal complexes (e.g. Zn-protein complexes); biodegradablepolymers such as polyesters; and/or salt-forming counterions such assodium.

The pharmaceutical compositions to be used for in vivo administrationmust be sterile. This is readily accomplished by filtration throughsterile filtration membranes, prior to, or following, preparation of thecomposition.

The pharmaceutical compositions comprising antibodies or bindingfragments thereof are for use in, but not limited to, diagnosing,detecting, or monitoring a condition mediated by an amyloidogenicprotein or peptide, and preventing, treating, managing, or amelioratinga condition, or one or more symptoms thereof, mediated by anamyloidogenic protein or peptide.

In one embodiment, the antibodies described herein, binding fragmentsthereof, or a pharmaceutical composition containing the same, areemployed in a method of prophylactically inhibiting the onset of acondition mediated by an amyloidogenic protein or peptide in a subject.This method involves administering to the subject the pharmaceuticalcomposition comprising an antibody or binding fragment thereof asdescribed herein, where the composition is administered in an amounteffective to prophylactically inhibit onset of the condition or one ormore of the symptoms of the condition mediated by an amyloidogenicprotein or peptide in the subject.

In another embodiment, the antibodies described herein, bindingfragments thereof, or a pharmaceutical composition containing the sameare employed in a method of treating a condition mediated by anamyloidogenic protein or peptide in a subject. This method involvesadministering to the subject a pharmaceutical composition comprising anantibody or binding fragment thereof as described herein, where thecomposition is administered in an amount effective to treat orameliorate the condition, or one or more symptoms thereof, mediated byan amyloidogenic protein or peptide in the subject.

In accordance with these embodiments, a condition mediated by anamyloidogenic protein includes, without limitation, Alzheimer's disease(AD) and all its variations, preclinical AD (i.e., individuals havingevidence of brain amyloid deposition with or without tau pathology (seeSperling et al., Alzheimer's & Dementia 7:280-92 (2011), which is herebyincorporated by reference in its entirety)), Rapid Progressive dementia,Down syndrome (DS), fronto-temporal dementia (FTD), Lewy Body Dementia(LBD), Parkinson's disease (PD), hereditary cerebral hemorrhage withamyloidosis (HCHWA), kuru, Creutzfeldt-Jakob disease (CJD)-familial,sporadic or new variant (nV)-, chronic wasting disease (CWD) and itsadapted forms in other mammals, Gerstmann-Straussler-Scheinker disease(GSS), Huntington's disease (HD) and all its glutamine expansionrepeats, fatal familial insomnia, British familial dementia and all itsvariations, Danish familial dementia and all its variations,frontotemporal lobar degeneration associated with protein tau(FTLD-tau), frontotemporal lobar degeneration associated with proteinFUS (FTLD-FUS), FTD-TDP-43, Amyotrophic lateral sclerosis (ALS), FTD andALS with all repeat expansions due to mutations on C9orf72, MildCognitive Impairment (MCI), familial corneal amyloidosis, Familialcorneal dystrophies, medullary thyroid carcinoma, insulinoma, type 2diabetes, isolated atrial amyloidosis, pituitary amyloidosis, aorticamyloidosis, plasma cell disorders, familial amyloidosis, senile cardiacamyloidosis, inflammation-associated amyloidosis, familial Mediterraneanfever (FMF), dialysis-associated amyloidosis, systemic amyloidosis,familial systemic amyloidosis, motor neuron disease, traumatic braininjury (TBI), and chronic traumatic encephalopathy, bovine spongiformencephalopathy (BSE) and its adapted forms in other mammals, ovineScrapie (Sc) and its adapted forms in other mammals.

In another embodiment, the antibodies described herein, bindingfragments thereof, or a pharmaceutical composition containing the sameare employed in a method of treating a subject having or at risk ofhaving a condition mediated by a pathological protein having a β-sheetsecondary structure. This method involves administering to the subject apharmaceutical composition comprising an antibody or binding fragmentthereof as described herein, where the composition is administered in anamount effective to treat, inhibit, or ameliorate the condition, or oneor more symptoms thereof, mediated by the pathological protein havingthe β-sheet secondary structure.

Conditions associated with one or more pathological proteins having aβ-sheet secondary structure that are suitable for treatment with theantibodies and binding fragments described herein include, withoutlimitation, certain cancers (i.e. cancer cells which produce proteins inhigh concentration that are prone to be misfolded and express a β-sheetsecondary structure), septic or toxic shock and associated inflammation(i.e. where protein fragments in cells oligomerize to form pores andexpress a β-sheet secondary structure), acquired immunodeficiencysyndrome (AIDS) (i.e., gp120), necrotic autoimmune reaction, andinfluenza (i.e. by targeting the hemagglutinin stalk structure (seeWilson et al., Nature 289:366-373 (1981), which is hereby incorporatedby reference in its entirety)).

In accordance with these embodiments, the “subject” is typically ahuman, but in some diseases, such as prion protein related diseases, thesubject can be a non-human mammal, such as a bovine. Other non-humanmammals amenable to treatment in accordance with the methods describedherein include, without limitation, primates, dogs, cats, rodents (e.g.,mouse, rat, guinea pig), camelids, horses, deer, cervids, cattle andcows, sheep, all ungulates, and pigs.

In prophylactic applications, the pharmaceutical compositions of thepresent invention are administered to a subject that is susceptible to,or otherwise at risk of, a particular condition mediated by anamyloidogenic protein or peptide in an amount sufficient to eliminate orreduce the risk of the condition or to delay, inhibit, or prevent theonset of the condition. Prophylactic application also includes theadministration of an antibody composition to prevent or delay therecurrence or relapse of a condition mediated by an amyloidogenicprotein or peptide. In the case of Alzheimer's disease, for example,virtually anyone is at risk of suffering from Alzheimer's disease if heor she lives long enough. Therefore, the compositions of the presentinvention can be administered prophylactically to the general populationwithout the need for any assessment of the risk of the subject patient.The present methods and compositions are especially suitable forprophylactic treatment of individuals who have a known genetic risk ofAlzheimer's disease or other condition related to an amyloidogenicprotein. Genetic markers associated with a risk of Alzheimer's diseaseinclude mutations in the APP gene, particularly mutations at position717 and positions 670 and 671 referred to as the Hardy and Swedishmutations respectively. Other markers of risk are mutations in thepresenilin genes (PS1 or PS2), presence of the ApoE4 genotype, presenceof TREM2 genotypes associated with AD (such as the R47H, D87N, or H157Ypolymorphisms), family history of AD, hypercholesterolemia, oratherosclerosis.

In therapeutic applications, pharmaceutical compositions areadministered to a subject suspected of, or already suffering from anamyloidogenic condition in an amount sufficient to cure, or at leastpartially arrest or alleviate, one or more symptoms of the condition andits complications. An amount adequate to accomplish this is defined as atherapeutically- or pharmaceutically-effective dose. In bothprophylactic and therapeutic regimes, agents are usually administered inseveral dosages until a sufficient response has been achieved. Aneffective dose of the composition of the present invention, for thetreatment of the above described conditions will vary depending uponmany different factors, including means of administration, target site,physiological state of the patient, whether the patient is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic.

In accordance with the prophylactic and therapeutic methods describedherein, compositions comprising the antibody or binding fragmentsthereof are administered in a dosage ranging from about 0.0001 to 100mg/kg, and more usually 0.01 to 10 mg/kg of the recipient's body weight.For example, the antibody or binding fragment thereof is administered ina dosage of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/kg, or higher, forexample 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, or 100 mg/kg. An exemplary treatment regime entails administrationonce per every two weeks or once a month or once every 3 to 6 months. Insome methods, two or more antibodies with different bindingspecificities are administered simultaneously, in which case the dosageof each antibody administered falls within the ranges indicated.Intervals between single dosages can be weekly, monthly or yearly.Intervals can also be irregular as indicated by measuring blood levelsof antibody in the patient. Alternatively, antibody can be administeredas a sustained release formulation, in which case less frequentadministration is required. Dosage and frequency vary depending on thehalf-life of the antibody in the patient. In general, human antibodiesshow the longest half life, followed by humanized antibodies, chimericantibodies, and nonhuman antibodies. The dosage and frequency ofadministration can vary depending on whether the treatment isprophylactic or therapeutic. In prophylactic applications, a relativelylow dosage is administered at relatively infrequent intervals over along period of time. Some patients continue to receive treatment for therest of their lives. In therapeutic applications, a relatively highdosage at relatively short intervals is sometimes required untilprogression of the disease is reduced or terminated, and preferablyuntil the patient shows partial or complete amelioration of symptoms ofdisease. Thereafter, the patient can be administered a prophylacticregime.

The mode of administration of the antibody, binding fragment thereof, orpharmaceutical composition described herein may be any suitable routethat delivers the compositions to the host, such as parenteraladministration, e.g., intradermal, intramuscular, intraperitoneal,intravenous or subcutaneous, pulmonary; transmucosal (oral, intranasal,intravaginal, rectal); using a formulation in a tablet, capsule,solution, powder, gel, particle; and contained in a syringe, animplanted device, osmotic pump, cartridge, micropump; or other meansappreciated by the skilled artisan, as well known in the art. Sitespecific administration may be achieved by, for example, intrarticular,intrabronchial, intraabdominal, intracapsular, intracartilaginous,intracavitary, intracelial, intracerebellar, intracerebroventricular,intracolic, intracervical, intragastric, intrahepatic, intracardial,intraosteal, intrapelvic, intrapericardiac, intraperitoneal,intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal,intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine,intravascular, intravesical, intralesional, vaginal, rectal, buccal,sublingual, intranasal, or transdermal delivery.

Administration can be systemic or local. In one embodiment, it may bedesirable to administer the antibodies of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion, by injection, or by means of animplant, said implant being of a porous or non-porous material,including membranes and matrices, such as sialastic membranes, polymers,fibrous matrices (e.g., Tissuel®), or collagen matrices.

In another embodiment, compositions containing the antibody or bindingfragment thereof are delivered in a controlled release or sustainedrelease system. In one embodiment, a pump is used to achieve controlledor sustained release. In another embodiment, polymeric materials can beused to achieve controlled or sustained release of the antibodycompositions described herein. Examples of polymers used in sustainedrelease formulations include, but are not limited to, poly(2-hydroxyethyl methacry-late), poly(methyl methacrylate), poly(acrylic acid),poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides(PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol),polyacrylamide, poly(ethylene glycol), polylactides (PLA),poly(lactide-co-glycolides) (PLGA), and polyorthoesters. The polymerused in a sustained release formulation is preferably inert, free ofleachable impurities, stable on storage, sterile, and biodegradable.Also, the use of liposomes, microcapsules or microspheres, inclusioncomplexes, or other types of carriers known in the art are alsocontemplated.

In yet another embodiment, a controlled or sustained release system canbe placed in proximity of the prophylactic or therapeutic target, thusrequiring only a fraction of the systemic dose. Controlled and/orrelease systems for delivery of antibodies known in the art are suitablefor use and delivery of compositions containing the antibodies andbinding fragments thereof as described herein, see e.g., Song et al,“Antibody Mediated Lung Targeting of Long-Circulating Emulsions,” PDAJournal of Pharmaceutical Science & Technology 50:372-397 (1995); Cleeket al, “Biodegradable Polymeric Carriers for a bFGF Antibody forCardiovascular Application,” Pro. Intl. Symp. Control. Rel. Bioact.Mater. 24:853-854 (1997); and Lam et al., “Microencapsulation ofRecombinant Humanized Monoclonal Antibody for Local Delivery,” Proc.Intl. Symp. Control Rel. Bioact. Mater. 24:759-760 (1997), each of whichis incorporated herein by reference in their entireties.

In embodiments where the pharmaceutical composition comprisespolynucleotides encoding the antibody or binding fragment thereof asdescribed herein, the nucleic acid can be administered in vivo topromote expression of its encoded antibody, by constructing it as partof an appropriate nucleic acid expression vector and administering it sothat it becomes intracellular, e.g., by use of a retroviral vector (seee.g., U.S. Pat. No. 4,980,286 to Morgan et al., which is herebyincorporated by reference in its entirety), or by direct injection, orby use of microparticle bombardment (see e.g., a gene gun; Biolistic,Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, or by administering it in linkage to ahomeobox-like peptide which is known to enter the nucleus (see e.g.,Joliot et al, Proc. Natl. Acad. Sci. USA 88: 1864-1868 (1991), which ishereby incorporated by reference in its entirety). Alternatively, anucleic acid can be introduced intracellularly and incorporated withinhost cell DNA for expression by homologous recombination.

The polynucleotide compositions can result in the generation of theantibody in the subject within at least about 1 hour, 2 hours, 3 hours,4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11hours, 12 hours, 13 hours, 14 hours, 15 hours, 20 hours, 25 hours, 30hours, 35 hours, 40 hours, 45 hours, 50 hours, or 60 hours ofadministration of the composition to the subject. The composition canresult in generation of the synthetic antibody in the subject within atleast about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8days, 9 days, or 10 days of administration of the composition to thesubject. The composition can result in generation of the antibody in thesubject within about 1 hour to about 6 days, about 1 hour to about 5days, about 1 hour to about 4 days, about 1 hour to about 3 days, about1 hour to about 2 days, about 1 hour to about 1 day, about 1 hour toabout 72 hours, about 1 hour to about 60 hours, about 1 hour to about 48hours, about 1 hour to about 36 hours, about 1 hour to about 24 hours,about 1 hour to about 12 hours, or about 1 hour to about 6 hours ofadministration of the composition to the subject.

The composition, when administered to the subject in need thereof, canresult in the persistent generation of the antibody in the subject. Thecomposition can result in the generation of the antibody in the subjectfor at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47days, 48 days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55days, 56 days, 57 days, 58 days, 59 days, or 60 days.

If the compositions of the invention are to be administered topically,the compositions can be formulated in the form of an ointment, cream,transdermal patch, lotion, gel, shampoo, spray, aerosol, solution,emulsion, or other form well-known to one of skill in the art. See,e.g., Remington's Pharmaceutical Sciences and Introduction toPharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa.(1995), which is hereby incorporated by reference in its entirety. Fornon-sprayable topical dosage forms, viscous to semisolid or solid formscomprising a carrier or one or more excipients compatible with topicalapplication and having a dynamic viscosity greater than water aretypically employed. Suitable formulations include, without limitation,solutions, suspensions, emulsions, creams, ointments, powders,liniments, salves, and the like, which are, if desired, sterilized ormixed with auxiliary agents (e.g., preservatives, stabilizers, wettingagents, buffers, or salts) for influencing various properties, such as,for example, osmotic pressure. Other suitable topical dosage formsinclude sprayable aerosol preparations wherein the active ingredient,for example in combination with a solid or liquid inert carrier, ispackaged in a mixture with a pressurized volatile (e.g., a gaseouspropellant, such as freon) or in a squeeze bottle.

If the methods described herein involve intranasal administration of theantibody composition, the composition can be formulated in an aerosolform, spray, mist or in the form of drops. In particular, prophylacticor therapeutic agents for use according to the present invention can beconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebulizer, with the use of a suitable propellant(e.g., dichlorodifluoromethane, trichloro-fluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridges(composed of, e.g., gelatin) for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

If the methods described herein involve oral administration of theantibody compositions described herein, the compositions can beformulated orally in the form of tablets, capsules, cachets, gelcaps,solutions, suspensions, and the like. Tablets or capsules can beprepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (e.g., pregelatinised maize starch,polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g.,lactose, microcrystalline cellulose, or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc, or silica); disintegrants(e.g., potato starch or sodium starch glycolate); or wetting agents(e.g., sodium lauryl sulphate). The tablets may be coated by methodswell-known in the art. Liquid preparations for oral administration maytake the form of, but not limited to, solutions, syrups or suspensions,or they may be presented as a dry product for constitution with water orother suitable vehicle before use. Such liquid preparations may beprepared by conventional means with pharmaceutically acceptableadditives such as suspending agents (e.g., sorbitol syrup, cellulosederivatives, or hydrogenated edible fats); emulsifying agents (e.g.,lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oilyesters, ethyl alcohol, or fractionated vegetable oils); andpreservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbicacid). The preparations may also contain buffer salts, flavoring,coloring, and sweetening agents as appropriate.

Formulations for injection may be presented in unit dosage form (e.g.,in ampoules or in multi-dose containers) with an added preservative. Thecompositions may take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and may contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient may be in powder form for constitution with a suitablevehicle (e.g., sterile pyrogen-free water) before use. The methods ofthe invention may additionally comprise of administration ofcompositions formulated as depot preparations. Such long actingformulations may be administered by implantation (e.g., subcutaneouslyor intramuscularly) or by intramuscular injection. Thus, for example,the compositions may be formulated with suitable polymeric orhydrophobic materials (e.g., as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives (e.g., as asparingly soluble salt).

The methods of the invention encompass administration of compositionsformulated as neutral or salt forms. Pharmaceutically acceptable saltsinclude those formed with anions such as those derived fromhydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., andthose formed with cations such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc.

Generally, the ingredients of compositions are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the mode of administration is infusion, compositioncan be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the mode of administrationis by injection, an ampoule of sterile water for injection or saline canbe provided so that the ingredients may be mixed prior toadministration.

The antibodies, binding fragments thereof, or pharmaceuticalcompositions containing the same can be packaged in hermetically sealedcontainers such as an ampoule or sachette indicating the quantity of theantibody or binding fragment thereof. In one embodiment, one or more ofthe antibodies, or pharmaceutical compositions of the invention issupplied as a dry sterilized lyophilized powder or water freeconcentrate in a hermetically sealed container and can be reconstituted(e.g., with water or saline) to the appropriate concentration foradministration to a subject. In one embodiment, one or more of theantibodies or pharmaceutical compositions of the invention is suppliedas a dry sterile lyophilized powder in a hermetically sealed containerat a unit dosage of at least 5 mg, for example at least 10 mg, at least15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg,at least 75 mg, or at least 100 mg. The lyophilized antibodies orpharmaceutical compositions of the invention should be stored at between2° C. and 8° C. in its original container and the antibodies, orpharmaceutical compositions of the invention should be administeredwithin 1 week, for example within 5 days, within 72 hours, within 48hours, within 24 hours, within 12 hours, within 6 hours, within 5 hours,within 3 hours, or within 1 hour after being reconstituted. In analternative embodiment, one or more of the antibodies or pharmaceuticalcompositions of the invention is supplied in liquid form in ahermetically sealed container indicating the quantity and concentrationof the antibody. In a further embodiment, the liquid form of theadministered composition is supplied in a hermetically sealed containerat least 0.25 mg/ml, for example at least 0.5 mg/ml, at least 1 mg/ml,at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10mg/ml, at least 15 mg/ml, at least 25 mg/ml, at least 50 mg/ml, at least75 mg/ml or at least 100 mg/ml. The liquid form should be stored atbetween 2° C. and 8° C. in its original container.

The antibodies and binding fragments described herein can beincorporated into a pharmaceutical composition suitable for parenteraladministration. In one aspect, antibodies will be prepared as aninjectable solution containing 0.1-250 mg/ml antibody. The injectablesolution can be composed of either a liquid or lyophilized dosage formin a flint or amber vial, ampule or pre-filled syringe. The buffer canbe L-histidine (1-50 mM), optimally 5-10 mM, at pH 5.0 to 7.0 (optimallypH 6.0). Other suitable buffers include but are not limited to, sodiumsuccinate, sodium citrate, sodium phosphate or potassium phosphate.Sodium chloride can be used to modify the tonicity of the solution at aconcentration of 0-300 mM (optimally 150 mM for a liquid dosage form).

Cryoprotectants can be included for a lyophilized dosage form,principally 0-10% sucrose (optimally 0.5-1.0%). Other suitablecryoprotectants include trehalose and lactose. Bulking agents can beincluded for a lyophilized dosage form, principally 1-10% mannitol(optimally 2-4%). Stabilizers can be used in both liquid and lyophilizeddosage forms, principally 1-50 mM L-Methionine (optimally 5-10 mM).Additional surfactants include but are not limited to polysorbate 20 andBRIJ surfactants. The pharmaceutical composition comprising theantibodies described herein prepared as an injectable solution forparenteral administration, can further comprise an agent useful as anadjuvant, such as those used to increase the absorption, or dispersionof the antibody. A particularly useful adjuvant is hyaluronidase, suchas Hylenex® (recombinant human hyaluronidase). Addition of hyaluronidasein the injectable solution improves human bioavailability followingparenteral administration, particularly subcutaneous administration. Italso allows for greater injection site volumes (i.e. greater than 1 ml)with less pain and discomfort, and minimum incidence of injection sitereactions (see WO 04/078140 to Bookbinder et al., and U.S. Patent Appl.Publication No. US2006104968 to Bookbinder et al., which are herebyincorporated herein by reference in their entirety).

In another embodiment, the antibodies and binding fragments thereof asdescribed herein are immobilized on a solid support. In one embodiment,the antibodies and binding fragments thereof are immobilized on a columnor membrane used in dialysis as described in U.S. Pat. No. 8,318,175 toFrangione et al., which is hereby incorporated by reference in itsentirety. In accordance with this embodiment, a subject having acondition mediated by an amyloidogenic protein or peptide is treated bydialyzing the subject's blood through the column and/or membranecontaining the bound antibodies or binding fragments thereof to removeamyloidogenic proteins from the subject's blood that are recognized bythe bound antibodies or binding fragments. Such dialyzing bloodtreatments are used to reduce or eliminate the presence of specifictoxic amyloidogenic proteins of peptides free flowing in a subject'splasma. Preferred methods of dialysis include, without limitation,hemodialysis, plasma exchange, plasma perfusion and hemofiltration. Thedialysis treatment can take place over a period of 2-3 hours (orlonger), and is repeated as necessary. Typically, dialysis is conductedevery 1-7 days for as long as the concentration of free amyloidogenicprotein or peptide in the subject's blood remains high, e.g., above0.1-0.5 ng/ml (10-50% of mean plasma level).

Suitable solid support material for immobilization of the antibodies andbinding fragments thereof includes, without limitation, nitrocellulose,cellulose, nylon, plastic, rubber, polyacrylamide, agarose,poly(vinylalcoholo-co-ethylene). The solid support material can beformed in a variety of shapes, including flat dialyzers, semi-permeablemembranes, semi permeable hollow fibers, coils, permeable spheres,dialysis membranes, and plasmapheresis filters, optionally using linkermolecules such as PEG (polyethelene glycol) to attach the ligand (asdisclosed in WO 00/74824 to Bristow, which is hereby incorporated byreference in its entirety). In a hemofiltration device, the solidsupports may be, for example, beads, plates, hollow filters, hollowfibers, or any combination thereof.

The antibodies and binding fragments described herein can also beemployed in a number of diagnostic, prognostic and researchapplications.

Another aspect of the present disclosure is directed to a method ofdiagnosing an amyloidogenic condition or disease in a subject. Thismethod involves detecting, in the subject, the presence of anamyloidogenic protein or peptide using a diagnostic reagent, wherein thediagnostic reagent comprises an antibody or binding fragment describedherein. The diagnosis of an amyloid disease in the subject is based onthe detection of an amyloidogenic protein or peptide in the subject.

Detecting the presence of an amyloidogenic protein or peptide in asubject using the antibodies or antibody fragments thereof as describedherein can be achieved by obtaining a biological sample from the subject(e.g., blood, urine, cerebral spinal fluid, ocular lacrimal secretion,saliva, feces, nasal brushings and tissue or organ biopsy), contactingthe biological sample with the diagnostic antibody reagent, anddetecting binding of the diagnostic antibody reagent to an amyloidogenicprotein or peptide if present in the sample from the subject. Assays forcarrying out the detection of an amyloidogenic protein/peptide in abiological sample using a diagnostic antibody are well known in the artand include, without limitation, ELISA, immunohistochemistry, SIMOA(single molecule array), and Western blot.

In accordance with this and other embodiments described herein, theantibody or binding fragments described herein are coupled to adetectable label. The label can be any detectable moiety known and usedin the art. Suitable labels include, without limitation, radioisotopesor radionuclides (e.g., ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I,¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm; fluorescent labels (e.g., FITC rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,luciferase, alkaline phosphatase); chemiluminescent markers; biotinylgroups; predetermined polypeptide epitopes recognized by a secondaryreporter (e.g., leucine zipper pair sequences, binding sites forsecondary antibodies, metal binding domains, epitope tags); and magneticagents, such as gadolinium chelates.

Detecting the presence of amyloidogenic proteins or peptides in asubject using the diagnostic antibody reagent of the present inventioncan also be achieved using in vivo imaging techniques. In vivo imaginginvolves administering to the subject the antibody or binding fragmentsthereof described herein, and detecting the binding of the antibody orbinding fragment thereof to the amyloidogenic protein in vivo.

Diagnostic antibodies or similar reagents can be administered byintravenous injection into the body of the patient, or directly into thebrain by intracranial injection or by drilling a hole through the skull.The dosage of antibody should be within the same ranges as for treatmentmethods. In accordance with this embodiment, the antibody or bindingfragment is coupled to an imaging agent to facilitate in vivo imaging.The imaging agent can be any agent known to one of skill in the art tobe useful for imaging, preferably being a medical imaging agent.Examples of medical imaging agents include, but are not limited to,single photon emission computed tomography (SPECT) agents, positronemission tomography (PET) agents, magnetic resonance imaging (MRI)agents, nuclear magnetic resonance imaging (NMR) agents, x-ray agents,optical agents (e.g., fluorophores, bioluminescent probes, near infrareddyes, quantum dots), ultrasound agents and neutron capture therapyagents, computer assisted tomography agents, two photon fluorescencemicroscopy imaging agents, and multi-photon microscopy imaging agents.Exemplary detectable markers include radioisotypes (e.g., ¹⁸F, ¹¹C, ¹³N,⁶⁴Cu, ¹²⁴I, ⁷⁶Br, ⁸²Rb, ⁶⁸Ga, ^(99m)Tc, ¹¹¹In, ²⁰¹Tl or ¹⁵O, which aresuitable for PET and/or SPECT use) and ultra-small superparamagneticparticles of iron oxide (USPIO) which are suitable for MRI.

Diagnosis of an amyloidogenic condition is performed by comparing thenumber, size, and/or intensity of detected amyloidogenicproteins/peptides in a sample from the subject or in the subject, tocorresponding baseline values. An appropriate baseline value can be theaverage level of amyloidogenic protein/peptide found in a population ofundiseased individuals. Alternatively, an appropriate baseline value maybe the level of amyloid protein deposition in the same subjectdetermined at an earlier time.

The diagnostic methods described herein can also be used to monitor asubject's response to therapy. In this embodiment, detection ofamyloidogenic proteins or peptides in the subject is determined prior tothe commencement of treatment. The level of amyloidogenic protein orpeptide in the subject at this timepoint is used as a baseline value. Atvarious times during the course of treatment the detection ofamyloidogenic protein/peptide is repeated, and the measured valuesthereafter compared with the baseline values. A decrease in valuesrelative to baseline signals a positive response to treatment.

A related aspect of the disclosure is directed to a method ofidentifying a subject's risk for developing a condition mediated by anamyloidogenic protein or peptide. This method involves detecting, in thesubject, the presence of an amyloidogenic protein or peptide using adiagnostic reagent comprising the antibody or binding fragment thereofdescribed herein, and identifying the subject's risk of developing acondition mediated by the amyloidogenic protein or peptide based on theresults of the detecting step.

Methods of detecting the presence of an amyloidogenic protein/peptide inthe subject or in a sample from the subject include the in vitro and invivo methods described supra. In one embodiment, the subject is notexhibiting any definitive signs or symptoms of an amyloidogeniccondition, and employment of this method serves as an early diagnostic.In another embodiment, the subject is not exhibiting any signs orsymptoms of an amyloidogenic conditions, but has a geneticpredisposition to a condition and employment off this method serves topredict the likelihood that the individual will develop theamyloidogenic condition in the future. In either embodiment, appropriatetherapeutic and/or prophylactic intervention can be employed, e.g.,administration of a therapeutic compositions containing the antibodiesor binding fragments thereof in an amount effective to slow or preventthe onset or progression of the amyloidogenic condition.

Another aspect of the present disclosure is directed to a diagnostic kitthat comprises the antibody or binding fragment thereof as describedherein and a detectable label.

A suitable detectable label is any moiety attached to an antibody or ananalyte to render the reaction between the antibody and the analytedetectable. A label can produce a signal that is detectable by visual orinstrumental means. Various labels include signal-producing substances,such as chromogens, fluorescent compounds, chemiluminescent compounds,radioactive compounds, and the like. Representative examples ofdetectable labels include moieties that produce light, e.g., acridiniumcompounds, and moieties that produce fluorescence, e.g., fluorescein. Inthis regard, the moiety itself may not be detectable, but becomesdetectable upon reaction with yet another moiety.

Other suitable detectable labels include radioactive labels (e.g., H, I,S, C, P, and P), enzymatic labels (e.g., horseradish peroxidase,alkaline peroxidase, glucose 6-phosphate dehydrogenase, and the like),chemiluminescent labels (e.g., acridinium esters, thioesters, orsulfonamides; luminol, isoluminol, phenanthridinium esters, and thelike), fluorescent labels (such as fluorescein (e.g., 5-fluorescein,6-carboxyfluorescein, 3′6-carboxyfiuorescein, 5(6)-carboxyfiuorescein,6-hexachloro-fluorescein, 6-tetrachlorofiuorescein, fluoresceinisothiocyanate, and the like)), rhodamine, phycobiliproteins,R-phycoerythrin, quantum dots (e.g., zinc sulfide-capped cadmiumselenide), a thermometric label, or an immuno-polymerase chain reactionlabel.

EXAMPLES Example 1—Production of Monoclonal Antibodies AgainstConformational β-Sheet Secondary Structures Common to Oligomeric Formsof Different Pathological Peptides and Proteins of VariousNeurodegenerative Diseases

Synthesis and Polymerization of 13-mer Bri Peptide (pBri). The procedurewas performed as previously published (Goñi et al, “ImmunomodulationTargeting Abnormal Conformation Reduces Pathology in a Mouse Model ofAlzheimer's Disease,” PLoS One 5(10): e13391 (2010), which is herebyincorporated by reference in its entirety). Briefly, the 13 residuepeptide that corresponds to the carboxyl terminus of ABri(Cys-Ser-Arg-Thr-Val-Lys-Lys-Asn-Ile-Ile-Glu-Glu-Asn; SEQ ID NO: 111)was synthetized on an ABI 430A peptide synthesizer (AME Bioscience,Chicago, Ill.) at the Keck peptide synthesis facility at YaleUniversity, CT. Mass spectroscopy of the lyophilized end-product wasused to verify the expected molecular weight.

To have a stable oligomeric conformation and make the 13 mer Bri peptideimmunogenic by itself, the synthetic peptide was subjected to controlledpolymerization using the following protocol: The peptide was dissolvedat 3 mg/ml, in 100 mM borate-150 mM NaCl (BBS), pH 7.4. Fresh 1%glutaraldehyde in BBS was added to the peptide to a final 5 mMglutaraldehyde concentration and incubated in an Eppendorf block shakerat 800 rpm and 56° C. for 16 hrs (Goñi et al., “ImmunomodulationTargeting Abnormal Protein Conformation Reduces Pathology in a MouseModel of Alzheimer's Disease” PLoS. ONE 5:e13391 (2010), and Goñi etal., “Immunomodulation Targeting both Aβ and tau Pathological ConformersAmeliorates Alzheimer's Disease Pathology in TgSwDl and 3×Tg mousemodels” Journal of Neuroinflammation 10:150 (2013), both of which arehereby incorporated by reference in their entirety). The solution wasthen quenched with 0.5 M glycine to make the solution 100 mM in glycine.After five minutes the solution was diluted 1:3 with BBS transferred toa dialysis membrane with a MWCO 2000 (Spectra Laboratories, Rockleigh,N.J.) and dialyzed extensively against 200 volumes and three changes of2 mM BBS at 4° C., aliquoted and lyophilized. To determine the degree ofaggregation the original monomeric ABri peptide and polymerized 13 merBri peptide (p13Bri) were electrophoresed on 12.5% (SDS)-polyacrylamideTris-tricine gels together with the low range Rainbow™ molecular weightmarkers (Amersham Biosciences, Piscataway, N.J.) under reducingconditions, then transferred onto nitrocellulose membranes and blottedagainst a specific rabbit polyclonal anti-Bri. For circular dichroismthe ABri and p13Bri at 0.25 mg/ml in saline were analyzed as previouslydescribed (Goñi et al., “Immunomodulation Targeting Abnormal ProteinConformation Reduces Pathology in a Mouse Model of Alzheimer's Disease”PLoS. ONE 5:e13391 (2010), which is hereby incorporated by reference inits entirety). For electron microscopy studies, the original andpolymerized Bri peptides were incubated at 1 mg/ml in 50 mMphosphate-150 mM NaCl (PBS) pH 7.4. Within 24 hours, 3 μl of each samplewere placed onto carbon coated 400 mesh Cu/Rh grid (Ted Pella Inc.,Redding, Calif.) and stained with 1% uranyl acetate in distilled water(Polysciences, Inc, Warrington, Pa.). Stained grids were examined underPhilips CM-12 electron microscope and photographed with a Gatan (4 k×2.7k) digital camera. The EMs were also repeated on the aged samples keptat room temperature (RT) for one, two and four weeks.

Immunization of CD-1 Mice. Immunization of the CD-1 mice and thesubsequent hybridoma production were performed at the Bi-InstitutionalAntibody and BioResource Core Facility of Memorial Sloan KetteringCancer Center and The Rockefeller University. All procedures wereapproved by the Institutional Animal Care and Use Committee protocol#97-03-009 and followed NIH standards. The p13Bri peptide was dissolvedin sterile saline and mixed 4:1 for the first two inoculations and 9:1for the remaining inoculations, with Aluminum Hydroxide (Alum) adjuvant(Brenntag Biosector, Denmark) or with the Ribi-like Sigma Adjuvantsystem (each vial containing 0.5 mg Monophosphoryl Lipid A fromSalmonella Minnesota and 0.5 mg synthetic Trehalose Dicorynomycolate in2% oil [squalene]-Tween® 80-water) (Sigma-Aldrich, St. Louis, Mo.).Animals were immunized as shown in Table 10 and Table 11. Mice receivedbi-weekly subcutaneous inoculations of 50 μg of the p13Bri andsubsequent inoculations were reduced to 20 μg of immunogen. Bleedingswere done 7 days after each inoculation starting after the secondinjection. Differential antibody titers to Aβ1-40 and 1-42 weredetermined by enzyme-linked immunosorbent analysis (ELISA); the plasmafor any bleeding was diluted 1:150 with 50 mM Tris-Saline pH 7.2, 0.1%Tween 20 (TBS-T) and incubated on Immulon 2HB 96-well (Thermo, Waltham,Mass.) microtiter ELISA plates pre-coated with either 50 ng/well ofAβ1-40 or Aβ1-42 in 50 mM ammonium bicarbonate solution pH 9.6preincubated at RT for 6 and 24-48 hs respectively, as previouslydescribed (Goñi et al., “Immunomodulation Targeting Abnormal ProteinConformation Reduces Pathology in a Mouse Model of Alzheimer's Disease”PLoS. ONE 5:e13391 (2010), which is hereby incorporated by reference inits entirety). Bound antibodies were detected with horseradishperoxidase-labeled goat anti-mouse IgG (H+L) (GE Healthcare UK) or goatanti-mouse IgM(μ) (KPL Gaithersburg, Md., USA). The color developingsubstrate was Tetramethyl benzidine (TMB) (Pierce, Rockford, Ill.) andthe readings were made at 450 nm. After the 7th inoculation, the M4mouse was rested for 45 days before being injected intravenously (i.v.)with 10 μg of p13Bri without adjuvant; the terminal bleeding wasperformed 4 days later, before harvesting the spleen for fusion. Theremaining animals were inoculated s. c. 5 more times, for a total oftwelve inoculations, with 20 μg of p13Bri, rested for two and a halfmonths at which time an i.v. injection of 10 μg of p13Bri with noadjuvant was given to all the animals. Terminal bleedings and spleenharvesting for fusion were performed 4 days later. The spleens of M1 andM2 and the spleens of M3 and M5 were combined for only two fusions.

TABLE 10 Protocol of Immunization of Mouse M4 with p13Bri for theProduction of β-sheet Secondary Structure Conformational AntibodiesCross-Reacting to Oligomeric Forms of Proteins and Peptides Found inNeurodegenerative Diseases. p13Bri Date of Antigen Antigen to DateInoculation Amount Adjuvant Route of Identification of Bleed (In days)(μg/animal) Ratio* Inoculation of Bleed (In days) — NONE n/a NONEPre-Immune −7 T0  0 50 4:1 s.c — — 14 50 4:1 s.c — — — — — —  T1| 21 2820 9:1 s.c — — — — — — T2 35 49 20 9:1 s.c — — — — — — T3 56 69 20 9:1s.c — — — — — — T4 76 91 20 9:1 s.c — — — — — — T5 98 119  20 9:1 s.c —— — — — — T6 126  165  10 no adjuvant i.v — — 169  Splenocytes — — TTBM4 169  fused to SP2/0-IL6** *Sigma adjuvant system SC: Subcutaneus: IV:Intravenous: **Fusion Partner: TTB: Terminal bleeding.

TABLE 11 Protocol for the Immunization of CD-1 Mice (M1 to M5) withp13Bri for the Production of β-sheet Secondary StructureAnti-Conformational Antibodies p13Bri Date of Antigen Antigen to DateInoculation Amount Adjuvant Route of Identification of Bleed (In days)(μg/animal)* Ratio** Inoculation of Bleed (In days) — — — — Pre-Immune −7 T0  0 50 4:1 s.c. — —  14 50 4:1 s.c. — — — — — — T1  21  28 20 9:1s.c. — — — — — — T2  35  49 20 9:1 s.c. — — — — — — T3  56  69 20 9:1s.c. — — — — — — T4  76  91 20 9:1 s.c. — — — — T5  98 119 20 9:1 s.c. —— — — T6 126 140 20 (no M4) 9:1 s.c. — — — — T7 147 161 20 (no M4) 9:1s.c. — — — — T8 166 165 10 (no M4) no adjuvant i.v. — — 169 M4 Fusion to— — terminal 169 SP2/0-IL6*** bleeding M4 181 20 9:1 s.c. — — — — T9 189201 20 9:1 s.c. — — — —  T10 208 258 20 9:1 s.c. — — — —  T11 265 332 10(M1, M2, no adjuvant i.v. — — M3, M5) 336 M1 + M2 Fused — — TTB M1, M2,336 SP2/0-IL6*** M3, M5 336 M3 + M5 Fused — — TTB M1, M2, 336SP2/0-IL6*** M3, M5 — — — — M1 + M2 Frozen 341 — — — — M3 + M5 Frozen341 *All animals inoculated unless indicated. **Alum M1, M2; Sigmaadjuvant system: M3, M4, M5. s.c.: subcutaneous, i.v.: introvenous. TTB:Terminal bleeding. ***Fusion partner

Monoclonal Production. Mouse M4 was sacrificed 165 days after the firstinoculation. The spleen was taken and splenocytes were gently dislodgedand fused to SP2/0-IL6 cells (ATCC® CRL-2016™) using Polyethylene Glycol1500 (Sigma-Aldrich St. Louis, Mo.). Fusion mixture was recoveredovernight at 3 million pre-fusion viable cells per ml. Half of thefusion was cryopreserved and from the other half cells were plated thefollowing day in a 96-well plate at 75,000 pre-fusion viable nucleatedsplenocytes per well at a final volume of 200 Ill/well. Cells werecultured for 7 days, then the cells were fed and after 3 days screeningsbegan. The media used was Gibco® Hybridoma-SFM (Fisher Scientific, USA);15% Fetal Bovine Serum, Hybridoma Fusion and Cloning supplement (HFCS)(Sigma-Aldrich, St. Louis, Mo.)—2× for the fusion and selection in HATand 1× during the screening-; 1× Gibco® HT Supplement (FisherScientific, Waltham, Mass.), 10 μg Gentamicin sulfate/ml (FisherScientific, Waltham, Mass.). The cloning protocol was a serial dilutiondone in a 96-well plate, and screening of wells with only one colony. Toassess for the presence of possible conformational antibodies in anystep of the screening and cloning, approximately 125 μL of cellsupernatants were diluted 1:1 with TBS-T and 50 μL/well applied toImmulon 2HB 96-well (Thermo, Waltham, Mass.) microtiter ELISA platespre-coated with either Aβ1-40, Aβ1-42, PrPRes or purified human pairedhelical filaments (PHF) from an AD subject, in a 50 mM ammoniumbicarbonate solution pH 9.6 as previously described (Goñi et al.,“Immunomodulation Targeting Abnormal Protein Conformation ReducesPathology in a Mouse Model of Alzheimer's Disease” PLoS. ONE 5:e13391(2010), and Goñi et al., “Immunomodulation Targeting both Aβ and tauPathological Conformers Ameliorates Alzheimer's Disease Pathology inTgSwDl and 3×Tg mouse models” Journal of Neuroinflammation 10:150(2013), both of which are hereby incorporated by reference in theirentirety). Bound antibodies were detected with horseradishperoxidase-labeled goat anti-mouse IgG+IgA+IgM(H+L) (KPL, Gaithersburg,Md., USA). The color developing substrate was Tetramethyl benzidine(TMB) (Pierce, Rockford, Ill.) and the readings were made at 450 nm.

Samples that were positive for more than one antigen (positivity beingdefined as a titer more than three times over the background) werecloned again using the same procedure, followed by testing after threedays growth. All clones positive for more than one antigen three timeswere cultured in 5 ml tubes until saturation. The tubes were centrifugedat 3,000×g for 10 minutes at 4° C.; supernatants were kept and thepelleted cells were divided into at least four vials containing 2×106cells in 0.5 mL of media diluted in half in DMSO, and cryopreserved inliquid nitrogen for storage and future expansion.

Mice M1, 2, 3 and 5 were sacrificed 332 days after the firstinoculation. The splenocytes of mice M1 and M2 and the splenocytes ofmice M3 and M5 were combined in equal ratios for two fusions as abovedescribed and cryopreserved for future use.

Partial Purification of Monoclonal Antibodies. Monoclonal antibodiespresent in the supernatants obtained after the fusion of the splenocytesof M4 CD-1 mouse and partner cells SP2/0-IL6, with cloning by serialdilutions, were partially purified by precipitation with SaturatedAmmonium Sulfate (SAS)—761.5 g/Lt at 21° C. (Berasain et al., “FasciolaHepatica: Parasite-Secreted Proteinases Degrade All Human IgGSubclasses: Determination of the Specific Cleavage Sites andIdentification of the Immunoglobulin Fragments Produced” Exp Parasitol94:99-110 (2000), which is hereby incorporated by reference in itsentirety). Samples were made 40% in the SAS, incubated at RT for atleast 4 hours, centrifuged at 14,000×g for 15 minutes, the supernatantseparated and the precipitate washed with a comparable volume of 40%SAS, centrifuged again and the supernatant pooled with the initialsupernatant. The precipitate was fractionated and kept at 4° C. untilfurther use. To assess the partial purification of the monoclonals andthe specific reactivity, aliquots were dissolved directly into tricinesample buffer (BioRad, Hercules Calif.) and electrophoresed at ˜1-2μg/lane. The samples for antibody activity were dissolved in distilleddeionized water (DDW) to half the original volume and subsequentlybrought to the desired dilution with the appropriate buffers for thetechnique.

Oligomerization of Neurodegenerative Antigens. Human tissue relatedstudies were performed under a protocol approved by the InstitutionalReview Board at New York University School of Medicine. In all cases,written informed consent for research was obtained from the patient orlegal guardian, and the material used had appropriate ethical approvalfor use in this project. All patients' data and samples were coded andhandled according to NIH guidelines to protect patients' identities.

Antigens known to be relevant in different neurodegenerative diseases,i.e. Aβ1-40 and Aβ1-42 (amyloidogenic in Alzheimer's Disease [AD] andother dementia), α-synuclein (Parkinson disease [PD] and Lewy BodyDementia), prion protein (PrPRes) (in prion disease) were polymerized tostable oligomeric states by the same glutaraldehyde methodology used toproduce the p13Bri (Goñi et al., “Immunomodulation Targeting AbnormalProtein Conformation Reduces Pathology in a Mouse Model of Alzheimer'sDisease” PLoS. ONE 5:e13391 (2010), and Goñi et al., “ImmunomodulationTargeting both Aβ and tau Pathological Conformers AmelioratesAlzheimer's Disease Pathology in TgSwDl and 3×Tg mouse models” Journalof Neuroinflammation 10:150 (2013), both of which are herebyincorporated by reference in their entirety). To produce stable fibrils1 mg/ml of either synthetic Aβ1-40, Aβ1-42 and α-synuclein peptides wereincubated in PBS pH 7.2 at 37° C. for at least 72 hours, until most ofthe peptide produced fibrils as determined by EM. Recombinant deer PrPwas incubated in 50 mM Tris buffer pH 7.4 to obtain aggregated species.Oligomeric/aggregated tau was obtained by purifying PHF from known casesof human Alzheimer's disease, who fulfilled the National Institute ofAging-Reagan criteria for AD, obtained from the Alzheimer Brain Bank ofthe Alzheimer's Disease Center at NYU, as previously described (Goñi etal., “Immunomodulation Targeting Abnormal Protein Conformation ReducesPathology in a Mouse Model of Alzheimer's Disease” PLoS. ONE 5:e13391(2010), and Wrzolek et al., “Immune Electron MicroscopicCharacterization of Monoclonal Antibodies to Alzheimer NeurofibrillaryTangles” Am J Pathol 141:343-355 (1992), which are both herebyincorporated by reference in their entirety). Briefly, 30 gm of frontalcortex was homogenized in 75 ml of 50 mM Tris-buffered saline (TBS), pH7.4 using an Ultra Turrox T25 tissue homogenizer (IKA Works, Inc;Staufen, Germany). 75 ml of 20% sarcosyl in H2O was added to the sampleand it was homogenized again. The homogenized material was centrifugedat 3,500 rpm in a Beckman GPR centrifuge and 6 ml aliquots of thesupernatant were each layered over 1 ml TBS/0.1% sulfobetaine 3-14(SB3-14) (Sigma-Aldrich, St Louis, Mo.) and centrifuged in an Optima Maxultracentrifuge at 75,000 rpm for 2 hours at 20° C. Each pellet wasresuspended by sonication in 1 ml of 10% NaCl in TBS/0.1% SB3-14followed by the addition of 6 ml of 10% NaCl in TBS/0.1% SB3-14, layeredover 1 ml of 20% sucrose in 10% NaCl TBS/0.1% SB3-14 and centrifuged at75,000 rpm for 1.5 hours at 20° C. The final pellets were resuspended inTBS by sonication prior to use. Purified PHF was also treated withproteinase K (Sigma-Aldrich, USA) to release oligomers from fibrils, at1:100 in PBS pH 7.2 for 30 minutes at 37° C., immediately quenched withphenylmethanesuphonyl fluoride (PMSF) and either immediately dissolvedin sample buffer for use in blots or frozen at −80° C. for future use.

Electron Microscopy. Electron microscopy images, using negativestaining, to assess the conformational states of monomeric, oligomericor fibrillar forms of the aggregated and polymerized ABri, p13Bri,Aβ1-40 and Aβ1-42, α-synuclein, PHF and PrP were done as previouslydescribed and were taken at the NYULMC OCS Microscopy Core (Goñi et al.,“Immunomodulation Targeting both Aβ and tau Pathological ConformersAmeliorates Alzheimer's Disease Pathology in TgSwDl and 3×Tg MouseModels” Journal of Neuroinflammation 10:150 (2013), which is herebyincorporated by reference in its entirety). Samples were diluted 1 mg/mlin PBS pH 7.4 and vortexed before 3 μl of each one were placed ontocarbon coated 400 mesh Cu/Rh grid (Ted Pella Inc., Redding, Calif.).Negative staining was performed using 1% uranyl acetate diluted indistilled water (Polysciences, Inc, Warrington, Pa.). Stained grids wereexamined under Philips CM-12 electron microscope and photographed with aGatan (4 k×2.7 k) digital camera. In most cases samples were kept forweeks or months to repeat the EMs at different times to follow thefibrillization or the stability of the oligomers.

Immunohistochemistry. Histology was performed on aged (>16 months old)3×Tg (Odo et al., “Triple-Transgenic Model of Alzheimer's Disease withPlaques and Tangles: Intracellular Abeta and Synaptic Dysfunction”Neuron 39:409-421 (2003), which is hereby incorporated by reference inits entirety) mouse brain sections with extensive Aβ and tau pathology;or formalin fixed paraffin embedded brain cortex sections of human AD,human age-matched controls and human young controls obtained from theAlzheimer Brain Bank of the Alzheimer's Disease Center at NYU. 40 μmmouse brains sections fixed with periodate-lysine-paraformaldehyde(PLP), kept in DMSO cryoprotectant, were washed three times for 5minutes with PBS pH 7.2 and twice for 15 min with 0.3% hydrogen peroxideto quench endogenous peroxidase activity. Sections were then blockedwith MOM kit Blocking solution (Vector Laboratories, Burlingame, Calif.)following the manufacture's protocol and incubated overnight withconformational hyperimmune 3×Tg or CD-1 (M4) mouse plasma diluted 1:300in MOM kit Diluent Solution. The following day, sections were washedthree times with PBS and incubated 1 hour with biotinylated anti-mouseIgG (H+L) or anti-mouse IgM (μ specific chain) antibodies (VectorLaboratories, Burlingame, Calif.) diluted in PBS 1:1000 followed by 1hour incubation of Vectastain® AB solution (Vector Laboratories,Burlingame, Calif.) as indicated by the manufacturer's protocol. Slideswere developed with 3,3-diaminobenzidine tetrahydrochloride with 2.5%nickel ammonium sulfate (Acros Organics, NJ) diluted in 0.2M sodiumacetate (NaAc) pH 6. The reaction was stopped by removal of the nickelsolution and extensively rinsing with 0.2M NaAc before stabilizing withPBS and further mounting on glass slides with Depex® Mounting Media(Electron Microscopy Sciences, Hatfield, Pa.).

Paraffin embedded human brain sections were dewaxed and rehydrated withsuccessive washes of xylene (2×5 minutes), 100% ethanol (2×5 minutes),95% ethanol (5 minutes), 70% ethanol (5 minutes) and PBS (5 minutes).Next, slides underwent antigen retrieval by boiling for 20 minutes in 10mM sodium citrate-0.05%-Tween20 pH 6.0. Sections were then washed withPBS (3×5 minutes), followed by 0.3% hydrogen peroxide washing, twice 15minutes each. Next slides were washed with PBS (3×5 minutes) and blocked1 hour at RT with 10% normal goat serum [NGS] (Thermo Scientific)-0.2%Triton X-100 (Sigma-Aldrich, St Louis, Mo.) in PBS. Slides wereincubated overnight with the plasmas of hyperimmune 3×Tg or CD-1 (M4)mice diluted 1:300 in 3% NGS-0.2% Triton X-100. Slides were then washedthree times with PBS and incubated for 1 hour with biotinylatedanti-mouse IgM (μ specific chain) antibody diluted 1:1000 in PBSfollowed by 1 hour incubation of Vectastain® AB solution. Slides werefurther developed with 3,3-diaminobenzidine tetrahydrochloride withnickel ammonium sulfate as described above.

To assess the reactivity of the hybridoma derived monoclonal antibodiesobtained after the fusion, Human brain sections were treated as abovedescribed but no antigen retrieval was performed. Monoclonal antibodies23B7, 12E9, 10E8 and 10F7 were used with a dilution of 1:2500 andmonoclonal antibody 3D7 was used at 1:3000.

For immunofluorescence, paraffin embedded human AD brain slides weredewaxed and rehydrated as described above, then blocked for 1 hour at RTwith 10% NGS-0.2% Triton X-100 in PBS and then incubated overnight at 4°C. with plasma of a CD-1 (M4) mouse hyperimmunized with p13pBri, diluted1:300 in PBS-T. Slides were then washed with PBS (3×5 minutes) andincubated 2 hours with Alexa Fluor® 488-conjugated goat anti-mouse IgMand Alexa Fluor® 647-conjugated goat anti-mouse IgG (JacksonImmunoResearch, West Groove, Pa.) both diluted 1:500 in PBS, followed by10 minutes incubation with bisBenzimide H 33342 trihydrochloride(Sigma-Aldrich, St. Louis, Mo.) diluted 1 μl/ml. Slides were then washedin PBS (3×5 minutes) and coverslipped with PermaFluor™ Aqueous MountingMedium (Thermo Scientific, Waltham, Mass.).

All slides were first screened on a Leica DM LB 100T microscope, thanscanned using a Hamamatsu Nanozoomer 2.0HT Digital Slide Scanner(Hamamatsu, Shizuoka Prefecture, Japan) at the NYU OCS ExperimentalPathology Histology Core. The images were viewed using the Slidepathsoftware (Leica, Wetzlar, Germany).

Electrophoresis and Western Blot. To characterize the monoclonalantibodies obtained after the fusion and cloning, 1 μg of each antibodywere mixed with an equal volume of tricine sample buffer,electrophoresed on Bolt™ 4-12% Bis-Tris gels and buffer (Thermo,Waltham, Mass.) under non-reducing conditions and transferred ontonitrocellulose membranes for 1 hour at 386 mA in 0.1%3-(Cyclohexylamino)-1-propanesulfonic acid (CAPS) (Sigma-Aldrich, St.Louis, Mo.)-10% methanol. To assess equal protein loading in each lane,membranes were stained for 1 min with reversible 0.1% Fast Green FCF(Sigma-Aldrich CO, USA), as per the manufacturer's instructions, in 25%methanol-10% Acetic Acid. The background was de-stained with rapidchanges of 25% methanol, followed by transfer to distilled water beforescanning on a Canon F916900 scanner Canon Inc, China). Membranes werethen washed in TBS-T for at least 15 minutes (until the reversible stainwas removed from the proteins on the membrane), blocked with 5% non-fatdry milk in TBS-T pH 8.3, for 1h at RT, washed three times with TBS-T,and then incubated 45 minutes with anti-mouse μ chain specific diluted1:8000 (KPL, Gaithersburg, Md.) or anti-mouse Kappa diluted 1:5000(Southern Biotech, Birmingham, Ala.). Bound antibodies were detectedwith the ECL detection system (Pierce, Rockford, Ill.) onautoradiography films (MIDSCI, St Louis, Mo.).

To determine the reactivity of the anti-conformational monoclonalantibodies to Aβ1-40, Aβ1-42, α-synuclein, PHF, PrP^(Res) 22L, sheepscrapie and deer PrP, each peptide or protein was loaded 1-2 μg/lane andelectrophoresed on Bolt™ 4-12% Bis-Tris gels (Thermo, Waltham, Mass.)under non-reducing conditions using 3 μl of High range Rainbow™molecular weight marker (Amersham Biosciences, Piscataway, N.J.) andlater transferred onto nitrocellulose membranes for 1 hour at 386 mA in0.1% CAPS-10% methanol. To assess the protein loading in each lane, themembranes were first stained with reversible 0.1% Fast Green FCF asdescribed above. Membranes were scanned before being blocked with 5%non-fat dry milk in TBS-T pH 8.3, for 1h at RT and washed three timeswith TBS-T. Membranes were then incubated with each monoclonal antibody,diluted 1:750 in TBS-T, or monoclonal anti-Aβ antibodies 4G8/6E10(1:4000) (BioLegend, San Diego, Calif.), monoclonal anti-α-synucleinAb-2 (1:3000) (Thermo, Waltham, Mass.), PHF-1 (1:2000) (which recognizesphosphorylated serine in positions 396 and 404, kindly provided by Dr.Peter Davies from the Feinstein Institute for Medical Research,Manhasset, N.Y.) or anti-PrP antibodies 7D9/6D11 (1:8000) (BioLegend,San Diego, Calif.).

Membranes were incubated later with peroxidase-linked anti-mouse IgG (GEHealthcare UK) (1:4000) for anti-α-synuclein, PHF-1 and 7D9/6D11. Todetect bound monoclonal antibodies anti-mouse p chain specific was useddiluted at 1:8000 (KPL, Gaithersburg, Md.).

Results. The ABri peptide selected as an immunogen is only 13 aminoacids long with no sequence homology to any known mammalian protein. Itcan adopt a β-sheet secondary structure and, by aggregation, formprotofibrils evolving into an amyloid fibrillar form (FIG. 1B, lowerpathway and FIGS. 2A-2D). To avoid fibril formation that previouslycomplicated the immune response in humans, glutaraldehyde was selectedfor cross-linking polymerization. The ABri has two preferential Lysylresidues at positions 6 and 7 amenable to the covalent linkage of morethan one unit through a glutaraldehyde bridge (Goñi et al.,“Immunomodulation Targeting Abnormal Protein Conformation ReducesPathology in a Mouse Model of Alzheimer's Disease” PLoS. ONE 5:e13391(2010), and Goñi et al., “Immunomodulation Targeting both Aβ and tauPathological Conformers Ameliorates Alzheimer's Disease Pathology inTgSwDl and 3×Tg Mouse Models” Journal of Neuroinflammation 10:150(2013), which are both hereby incorporated by reference in theirentirety). However, the free NH₂ group from the amino-terminus of a verysmall peptide, or the two adjacent Lysyl residues could be prone to forma Schiff base with the glutaraldehyde preventing further association(Moore et al., “Biophysical Analyses of Synthetic Amyloid-Beta(1-42)Aggregates Before and After Covalent Cross-Linking. Implications forDeducing the Structure of Endogenous Amyloid-Beta Oligomers”Biochemistry 48:11796-11806 (2009), and Migneault et al.,“Glutaraldehyde: Behavior in Aqueous Solution, Reaction with Proteins,and Application to Enzyme Crosslinking” Biotechniques 37:790-796,798-802 (2004), which are both hereby incorporated by reference in theirentirety). Thus, a mild alkaline pH was selected for the reaction tostabilize the lysines net charge and maximize separation by chargerepulsion, a high temperature and high rpm shaking was utilized to avoidstabilizing a blocked monomeric structure during the chemical reaction,a buffer with no phosphate or Tris groups that could interfere with theprogression of oligomerization was selected, and a glutaraldehydeconcentration to equalize the ratio of long self-polymerizedglutaraldehyde chains versus the joining of two or more ABri peptideswas selected (Goñi et al., “Immunomodulation Targeting Abnormal ProteinConformation Reduces Pathology in a Mouse Model of Alzheimer's Disease”PLoS. ONE 5:e13391 (2010), Goñi et al., “Immunomodulation Targeting bothA β and tau Pathological Conformers Ameliorates Alzheimer's DiseasePathology in TgSwDl and 3×Tg Mouse Models” Journal of Neuroinflammation10:150 (2013), and Migneault et al., “Glutaraldehyde: Behavior inAqueous Solution, Reaction with Proteins, and Application to EnzymeCrosslinking” Biotechniques 37:790-796, 798-802 (2004), which are eachhereby incorporated by reference in their entirety). Nevertheless, somemonomers, dimers and trimers were formed as detected in gels by proteinstain or specific antisera (FIG. 1B, green pathway, EM, CD and blot ofFIGS. 2A-2D). These lower molecular weight forms still consisted of apredominant β-sheet secondary structure, but never aggregate intofibrils. The rest of the covalently linked oligomers distributed between10 and 100 kDa, remained stable for very long periods of time, neverforming potentially cross-seeding fibrils. This resulting p13Briimmunogen is composed from many intermediate size covalently linkedoligomers with a high number of repetitions of the small 13 mer motif ina predominant β-sheet secondary structure (FIGS. 1A-1C and FIGS. 2A-2D).

The previously published p13Bri vaccine inoculated with AluminumHydroxide (Alum) as an adjuvant produced a mild polyclonal response topathologic oligomeric forms present in three mouse models of AD; i.e.,Tg APP/PS1 (with mainly amyloid plaques), Tg SwDI (with extensivevascular amyloid), and 3×Tg APP/PS1 P301L (with combined Aβ and taupathologies) (Wisniewski et al. “Immunotherapeutic Approaches forAlzheimer's Disease” Nueron 85:1162-1176 (2015), Goni et al.,“Immunomodulation Targeting Abnormal Protein Conformation ReducesPathology in a Mouse Model of Alzheimer's Disease” PLoS ONE 5:e13391(2010), and Goñi et al., “Immunomodulation Targeting both A β and tauPathological Conformers Ameliorates Alzheimer's Disease Pathology inTgSwDl and 3×Tg Mouse Models” Journal of Neuroinflammation 10:150(2013), which are each hereby incorporated by reference in theirentirety) that most probably was not enough to obtain sufficient B-cellswith the desired paratopes of the monoclonal antibodies obtained herein.Nevertheless, for the prevention therapy the small polyclonal responsewas sufficient as proved in all three AD mouse models, where ADpathology was greatly reduced and cognitive rescue was achieved by earlyvaccination (Wisniewski et al. “Immunotherapeutic Approaches forAlzheimer's Disease” Neuron 85:1162-1176 (2015), Goñi et al.,“Immunomodulation Targeting Abnormal Protein Conformation ReducesPathology in a Mouse Model of Alzheimer's Disease” PLoS. ONE 5:e13391(2010), and Goñi et al., “Immunomodulation Targeting both A (3 and tauPathological Conformers Ameliorates Alzheimer's Disease Pathology inTgSwDl and 3×Tg Mouse Models” Journal of Neuroinflammation 10:150(2013), which are each hereby incorporated by reference in theirentirety). To reproduce the successful immune response, increase thesustainable antibody concentration for monoclonal production purposes,and avoid possible interference from the transgenes of the AD mousemodels, 5 wild type CD-1 mice were inoculated with a modified protocol,including some with a RiBi-like Sigma adjuvant to enhance the antibodyimmune response (See Table 11).

The elicited polyclonal antibody response was analyzed by ELISA withdifferential Aβ1-40 and Aβ1-42 coats known to develop β-sheetaggregation (Goñi et al., “Immunomodulation Targeting Abnormal ProteinConformation Reduces Pathology in a Mouse Model of Alzheimer's Disease”PLoS. ONE 5:e13391 (2010), and Goñi et al., “Immunomodulation Targetingboth Aβ and tau Pathological Conformers Ameliorates Alzheimer's DiseasePathology in TgSwDl and 3×Tg Mouse Models” Journal of Neuroinflammation10:150 (2013), which are both hereby incorporated by reference in theirentirety). Each Aβ peptide was dissolved in bicarbonate buffer pH 9.6and left at RT to age; the Aβ1-40 for a few hours before coating and theAβ1-42 for at least two days before coating; enough in both cases toestablish aggregation and fibrillization. EM analysis of both Aβpeptides after aging, documented fibril formation; however, because ofthe high pH, a significant number of stable oligomeric forms surroundedthe fibrils of Aβ1-42 at a higher concentration per area than with theAβ1-40 fibrils (FIG. 3C). Even after using a completely differentadjuvant and a strong regime of inoculations (shown on Table 10 and 11)the antibody quality of the polyclonal IgM and IgG responses obtained inCD-1 animals were similar to the ones reported in the 3×Tg inoculatedanimals but more robust, raising the possibility of having enoughB-cells with the desired paratopes that could be derived into stablehybridoma cells (Goñi et al., “Immunomodulation Targeting both A β andtau Pathological Conformers Ameliorates Alzheimer's Disease Pathology inTgSwDl and 3×Tg Mouse Models” Journal of Neuroinflammation 10:150(2013), which is hereby incorporated by reference in its entirety), andcould distinguish the misfolded peptides and the differentialconcentration of oligomeric forms in both of them, with a much highertiter against Aβ1-42 versus Aβ1-40 (FIG. 3C and FIGS. 4A-4B). Thecontrol to assess for equal coating of both Aβ peptides was determinedwith two commercial IgG anti-Aβ primary structure monoclonal antibodies(mAbs) 4G8 and 6E10, that have similar reactivity for both Aβ 1-40 andAβ1-42 (FIG. 4C), demonstrating the differential data obtained fromp13Bri immunized animals depended on the recognition of misfoldedoligomeric forms (present at a higher concentration in the Aβ1-42preparation) rather than an unspecific cross-reaction to the primarystructure of the peptides used for plate coating.

By immunohistochemistry the plasma of the CD-1 inoculated animals aswell as the previously reported pooled plasma from successfullyvaccinated animals recognized similar neuronal cytoplasmic andextracellular material in the cerebral cortex and hippocampus ofuntreated old 3×Tg mice with extensive amyloid-β and tau pathologies(FIG. 3A) (Goñi et al., “Immunomodulation Targeting Abnormal ProteinConformation Reduces Pathology in a Mouse Model of Alzheimer's Disease”PLoS. ONE 5:e13391 (2010), and Goñi et al., “Immunomodulation Targetingboth A β and tau Pathological Conformers Ameliorates Alzheimer's DiseasePathology in TgSwDl and 3×Tg Mouse Models” Journal of Neuroinflammation10:150 (2013), which are both hereby incorporated by reference in theirentirety). The same plasma samples were used to immunolabel the temporalcortex of human AD brains. Both the plasma of previously vaccinatedanimals (Goñi et al., “Immunomodulation Targeting Abnormal ProteinConformation Reduces Pathology in a Mouse Model of Alzheimer's Disease”PLoS. ONE 5:e13391 (2010), and Goñi et al., “Immunomodulation Targetingboth A β and tau Pathological Conformers Ameliorates Alzheimer's DiseasePathology in TgSwDl and 3×Tg Mouse Models” Journal of Neuroinflammation10:150 (2013), which are both hereby incorporated by reference in theirentirety) and the plasma from p13Bri immunized CD-1 mice recognizedcomparable intracellular and extracellular pathologic features in humanAD sections but showed no significant immunolabeling in the cerebralcortex of human control brains with no AD pathology (FIG. 3B). Both IgMand IgG antibodies from the CD-1 animals co-localized with the samepathological structures in human AD brains (FIG. 5A-5B).

To test the feasibility of producing hybridomas with specificanti-β-sheet secondary structure, the CD-1 animal M4 that had the bestIgM and IgG polyclonal response to β-sheet oligomers was selected (FIGS.3A-3C and FIGS. 4A-4C). The protocol of the M4 p13Bri inoculations andfusion used for subsequent hybridoma production is shown in Table 10 andis described in herein.

After fusion of the M4 mouse spleen cells to the SP2/0-IL6 partnercells, the fused cells and the viable hybridomas were selected byincubation and serial dilution as per Table 10 and the MonoclonalProduction method described herein.

The anti-β-sheet secondary structure selection process was novel formonoclonal production and involved the simultaneous detection ofreactivity of plated viable hybridomas with four different NDDconformers. The small availability, in each round, of cell supernatantfrom three days growth of a limited number of hybridoma cells in 96 wellplates required a sensitive ELISA differential analysis. Theconcentration of Aβ oligomers around fibrils of Aβ1-40 and Aβ1-42samples was described above (FIG. 3C) and required two separate ELISAplates for analysis. Two additional ELISA plates were added, one platedwith PHF extracted from a human AD brain and treated to maximize thenumber of clusters of β-sheet oligomeric conformers around fibrils(FIGS. 6A and 7A) that are expected to be associated with β-sheet stericzippers characteristic of toxic oligomerization before they becomeburied in fibril structures (Sawaya et al., “Atomic Structures ofAmyloid Cross-Beta Spines Reveal Varied Steric Zippers” Nature447:453-457 (2007), and Avila et al., “Tau Structures” Front AgingNeurosci 8:262 (2016), which are both hereby incorporated by referencein their entirety). The other ELISA was plated with aged elk recombinantPrP produced in E. coli, which has properties resembling PrP^(Res) witholigomerization and exposed β-sheet motifs, without the extended β-sheetstructure characteristic of amyloidogenic and infectious PrP^(Sc) (FIGS.6B, 7A, and 7D) (Cobb et al., “Conformational Stability of MammalianPrion Protein Amyloid Fibrils is Dictated by a Packing PolymorphismWithin the Core Region” J Biol Chem 289:2643-2650 (2014), Ostapchenko etal., “Two Amyloid States of the Prion Protein Display SignificantlyDifferent Folding Patterns” J Mol Biol 400:908-921 (2010), Lauren etal., “Cellular Prion Protein Mediates Impairment of Synaptic Plasticityby Amyloid-Beta Oligomers” Nature 457:1128-1132 (2009), and Wiltzius etal., “Molecular Mechanisms for Protein-Encoded Inheritance” Nat StructMol Biol 16:973-978 (2009), each of which is hereby incorporated byreference in its entirety).

In the first round of selection, more than fifty limited volumesupernatants from samples of wells containing a few cells were analyzedat the same time precluding a further dilution for duplicates or forindividual plates to assess IgG and IgM classes, separately. Apolyclonal anti-mouse GAM, at a suitable dilution, was used to maximizedetection. As a positive control, commercial antibodies to each specificsequence conformer on the corresponding plates were used, assuring thehomogeneity of the coating. At the same time the anti-mouse GAM was ableto recognize IgM or IgG hybridomas producing readings of at least threetimes over the background as shown by comparison to irrelevant clones(FIGS. 7A, 8A, and 8B). Cells from all supernatants that were positivewith at least two of the four conformational selectors were subcloned asdescribed supra. The clones that were positive for at least twoconformers and maintained the reactivity for three rounds of subcloningwere deemed potential anti-β-sheet monoclonals, separated and expanded(FIG. 1C, blue pathway).

Thirty five potential clones were obtained using the above criteria,which could be divided into six families of similarly reactingmonoclonals from which the best representatives were 23B7; 10E8; 3D7;12E1; 11F2 and 10F7 (FIGS. 6B; 7A-7D, and 10A). All clones that werestable with a sustained production of an anti-β-sheet monoclonal werelater shown to be IgM-kappa in pentameric form (FIGS. 9A-9D), whereasunexpectedly, no stable IgG producing hybridomas survived the threerounds of selection.

After initial expansion of the potential anti-β-sheet secondarystructure clones, enough cell supernatant was available to corroboratethe ELISA reactivity in duplicate and was used as a source of primaryantibody in specific immunoblots that showed the poly-reactivity to thesame antigens that shared only a dominant β-sheet secondary structure,as well as to oligomerized α-synuclein. The immunoblots also showedspecific detection of low abundance oligomeric structures from every NDDconformer that were typically detected less well or not at all by theanti-primary structure dependent commercial antisera specific for onlyone type of protein or peptide (FIG. 6B).

To assure the anti-β-sheet conformation reactivity was due only to IgMmonoclonals; all six representative monoclonals were partially purifiedby ammonium sulfate precipitation (SAS) to remove more than 90% of theBSA and other contaminating proteins. The integrity of the IgMκ pentamerwas maintained as well as the antibody specificity (FIGS. 9A-9D).

Each one of the representative monoclonals reacted in gels in a specificway with the different NDD oligomeric conformers (FIGS. 7B-7D),including reactivity to polymerized recombinant α-synuclein aggregatedat the top of the gels, that has similarities to the toxic a synucleinoligomers that are found in PD and LBD (FIG. 7C). Each selectedmonoclonal shows evidence of cross reactivity, with reactivity to atleast two oligomeric conformers with differing primary sequence (FIGS.7B-7D).

The SAS semi-purified monoclonals were used to immunolabel the cortex ofhuman AD brains. Each monoclonal differentially labelled extracellularand cytoplasmic material: 23B7 strongly labels neuronal cytoplasmincluding processes and the nucleus; 3D7 labels the whole neuronal cellbody; with both labeling some extracellular material. 12E1preferentially labelled glial cells. 10E8 and 10F7 showed a lighterimmunolabeling of all neuronal cytoplasmic components (FIG. 10A). Noappreciable labelling was detectable with monoclonals using cortextissue of human control brains with no NDD pathology (FIG. 10B). Allreactivities can be traced as derived from the polyclonal response ofthe M4 mouse before fusion (FIGS. 3A, 3C, and FIG. 10A), demonstratingthe monoclonals were originally elicited by the p13Bri immunization andselected by using the different NDD conformers.

Discussion As described herein, using a novel methodology, anti-β-sheetsecondary structure monoclonal antibodies that recognize a dominantβ-sheet structure present in pathology associated oligomers of misfoldedprotein/peptides of different NDD have been developed.

The production of anti-β-sheet monoclonal antibodies to a particularsecondary structure present in oligomers of misfolded protein/peptidescan be achieved in a sequential manner that involves first production ofa stable oligomer preparation using a small non-self peptide polymerizedto itself. This polymerized peptide, referred to as p13Bri, is derivedfrom only the last 13 amino acids of the carboxyl terminus of the ABripeptide, oligomerized using glutaraldehyde as a cross linker to form astable population of sequence homogeneous oligomers, as previouslydescribed (Vidal et al., “A Stop-Codon Mutation in the BRI GeneAssociated with Familial British Dementia” Nature 399:776-781 (1999),and Rostagno et al., “Chromosome 13 Dementias” Cell Mol. Life Sci62:1814-1825 (2005), both of which are hereby incorporated by referencein their entirety) (FIGS. 1A-1C and 2A-2D). The carboxyl 13 residue endof ABri lacks any sequence homology to Aβ, tau or any other native humanproteins, since it is derived from an intronic transcript (Wisniewski etal., “Immunotherapeutic Approaches for Alzheimer's Disease” Expert RevVaccines 15:401-415 (2016), Vidal et al., “A Stop-Codon Mutation in theBR1 Gene Associated with Familial British Dementia” Nature 399:776-781(1999), Rostagno et al., “Chromosome 13 Dementias” Cell Mol. Life Sci62:1814-1825 (2005), and Goñi et al., “Immunomodulation TargetingAbnormal Protein Conformation Reduces Pathology in a Mouse Model ofAlzheimer's Disease” PLoS. One 5:e13391 (2010), each of which is herebyincorporated by reference in its entirety). The length of this specificsequence allows it to gain a dominant β-sheet secondary structure, butis too short for significant folding to a tertiary structure, whichwould introduce unwanted competing conformations in the resultingimmunogen. Thus, the polymerization process stabilized repeating motifswith only β-sheet secondary structure, increasing the oligomer size sothat it would be immunogenic by itself.

Prior work using 3 different AD transgenic (Tg) mouse models, has shownthat active immunization based on this approach produces a therapeuticpolyclonal response that reduces all three key neuropathologicalfeatures of AD, namely amyloid plaques, congophilic amyloid angiopathy(CAA), and tau related pathology, in association with significantcognitive benefits (Wisniewski et al., “Immunotherapeutic Approaches forAlzheimer's Disease” Neuron 85:1162-1176 (2015), Wisniewski et al.,“Developing Therapeutic Vaccines Against Alzheimer's Disease” Expert RevVaccines 15:401-415 (2016), Goñi et al., “Immunomodulation TargetingAbnormal Protein Conformation Reduces Pathology in a Mouse Model ofAlzheimer's Disease” PLoS. ONE 5:e13391 (2010), Goñi et al.,“Immunomodulation Targeting Both Aβ and tau Pathological ConformersAmeliorates Alzheimer's Disease Pathology in TgSwDl and 3×Tg MouseModels” Journal of Neuroinflammation 10:150 (2013), and Wisniewski andGoñi, “Immunotherapy for Alzheimer's Disease” Biochemical Pharmacology88:499-507 (2014), each of which is hereby incorporated by reference inits entirety). Amyloid plaques and CAA were shown to be reduced inAPP/PS1 (amyloid plaque model) and TgSwDI (CAA Tg model) model mice,respectively, while in 3×Tg mice (amyloid plaque and tau pathologymodel), p13Bri immunization led to reductions of both tau and Aβpathology (Wisniewski et al., “Immunotherapeutic Approaches forAlzheimer's Disease” Neuron 85:1162-1176 (2015), Wisniewski et al.,“Developing Therapeutic Vaccines Against Alzheimer's Disease” Expert RevVaccines 15:401-415 (2016), Goñi et al., “Immunomodulation TargetingAbnormal Protein Conformation Reduces Pathology in a Mouse Model ofAlzheimer's Disease” PLoS ONE 5:e13391 (2010), Goñi et al.,“Immunomodulation Targeting Both Aβ and tau Pathological ConformersAmeliorates Alzheimer's Disease Pathology in TgSwDl and 3×Tg MouseModels” Journal of Neuroinflammation 10:150 (2013), and Wisniewski andGoñi, “Immunotherapy for Alzheimer's Disease” Biochemical Pharmacology88:499-507 (2014), each of which is hereby incorporated by reference inits entirety). Inoculation of p13Bri with Alum as an adjuvant in thesethree AD Tg models produced a systemic polyclonal response topathologic/oligomeric forms of both Aβ and tau, with demonstratedcross-specificity to AD, prion disease, and LBD human brain tissue(FIGS. 3A-3C). These unusual results led to the production of hybridomasfrom which monoclonal antibodies with potential diagnostic ortherapeutic value were selected by their specific reactivity to β-sheetsecondary structures found in unrelated primary sequences of pathologicoligomeric conformers of diverse NDD.

In comparison to the previously reported successful active vaccinationusing p13Bri with Alum as an adjuvant in AD Tg mice, a longer and moreintense immunization protocol along with a RiBi-like adjuvant (FIGS.2A-2D) was employed herein to expand the antibody response to thedominant β-sheet secondary structure in oligomers and generate a greaternumber of spleen B-cells with anti-β-sheet receptors. Thus, the chanceof transforming these B-cells into antibody producing stable hybridomaswas increased. Thus, the polyclonal response was analyzed by ELISA testsas described herein with the understanding that detecting a differentialin Aβ oligomers concentration per area around fibrils may reflect moreaccurately the real biochemical dynamic around plaques in AD (Riek etal., “The Activities of Amyloids From a Structural Perspective” Nature539:227-235 (2016), which is hereby incorporated by reference in itsentirety). Plasma from the p13Bri immunized CD-1 animals after 6inoculations clearly showed a persistent IgM response to the oligomericforms of Aβ (FIG. 3C and FIGS. 4A-4C). These persistent polyclonal IgMs,similar to what was previously reported (Goñi et al., “ImmunomodulationTargeting Abnormal Protein Conformation Reduces Pathology in a MouseModel of Alzheimer's Disease” PLoS. ONE 5:e13391 (2010), Goñi et al.,“Immunomodulation Targeting Both Aβ and tau Pathological ConformersAmeliorates Alzheimer's Disease Pathology in TgSwDl and 3×Tg MouseModels” Journal of Neuroinflammation 10:150 (2013), and Sigurdsson etal., “An Attenuated Immune Response is Sufficient to Enhance Cognitionin an Alzheimer's Disease Mouse Model Immunized with Amyloid-βderivatives” J. Neurosci 24:6277-6282 (2004), each of which is herebyincorporated by reference in its entirety), were later shown to beconsistent and their IgM producing B-cells able to be transformed intostable monoclonals (FIGS. 6-10).

In order to assure detection and separation of only the clones withspecificity to a β-sheet secondary structure conformation, monoclonalclones were selected by testing reactivity to a number of differentoligomer preparations from various NDD that only share a common β-sheetsecondary structure, but no primary sequence or tertiary structurehomology. Clones were selected that had strong reactivity to at leasttwo distinct β-sheet secondary structure conformations, with a differingprimary sequence. The aged or oligomerized Aβ peptides used in ELISA andblots (FIGS. 6 and 7) reflect the known structures of bend parallel oranti-parallel β-sheet secondary structure, which will convert tooligomers and eventually fibrils (Sawaya et al, “Atomic Structures ofAmyloid Cross-Beta Spines Reveal Varied Steric Zippers” Nature447:453-457 (2007), Wiltzius et al., “Molecular Mechanisms forProtein-Encoded Inheritance” Nat Struct Mol Biol 16:973-978 (2009), andBarrow et al., “Solution Conformations and Aggregational Properties ofSynthetic Amyloid Beta-Peptides of Alzheimer's Disease. Analysis ofCircular Dichroism Spectra” J. Mol. Biol 225:1075-1093 (1992), each ofwhich is hereby incorporated by reference in its entirety). The PHFpurified from AD subjects and the PHF digested with proteinase K werespecific selectors of the dominant β-sheet secondary structureassociated with ptau toxic oligomers (FIGS. 6 and 7) (Walker et al.,“Mechanisms of Protein Seeding in Neurodegenerative Diseases” JAMANeurol 70:304-310 (2013), Sawaya et al., “Atomic Structures of AmyloidCross-Beta Spines Reveal Varied Steric Zippers” Nature 447:453-457(2007), Avila et al, “Tau Structures” Front Aging Neurosci 8:262 (2016),Wiltzius et al., “Molecular Mechanisms for Protein-Encoded Inheritance”Nat Struct Mol Biol 16:973-978 (2009), Daebel et al., “Beta-Sheet Coreof tau Paired Helical Filaments Revealed by Solid-State NMR” J Am ChemSoc 134:13982-13989 (2012), and von Bergen et al., “Assembly of tauProtein into Alzheimer Paired Helical Filaments Depends on a LocalSequence Motif ((306)VQIVYK(311)) Forming Beta Structure” Proc Natl AcadSci USA 97:5129-5134 (2000), which are each hereby incorporated byreference in their entirety), while the deer recombinant PrP served asan example of aggregation through β-sheet dominant motifs similar tothose found in oligomeric PrP^(Res) (Sawaya et al, “Atomic Structures ofAmyloid Cross-Beta Spines Reveal Varied Steric Zippers” Nature447:453-457 (2007), and Wiltzius et al, “Molecular Mechanisms forProtein-Encoded Inheritance” Nat Struct Mol Biol 16:973-978 (2009),which are both hereby incorporated by reference in their entirety). Inall cases, these new monoclonals recognized novel oligomeric structuresthat are not evident using conventional anti-primary structureantibodies (FIG. 6). These monoclonals are NDD pathology specific;however, each clone shows preferential binding to different oligomerspecies (FIGS. 7 and 10).

Many oligomer specific antibodies have been reported (Viola et al.,“Amyloid Beta Oligomers in Alzheimer's Disease Pathogenesis, Treatment,and Diagnosis” Acta Neuropathol 129:183-206 (2015), Kayed et al.,“Common Structure of Soluble Amyloid Oligomers Implies Common Mechanismof Pathogenesis” Science 300:486-489 (2003), Lee et al., “TargetingAmyloid-Beta Peptide (Abeta) Oligomers by Passive Immunization with aConformation-Selective Monoclonal Antibody Improves Learning and Memoryin Abeta Precursor Protein (APP) Transgenic Mice” J Biol Chem281:4292-4299 (2006), Tucker et al., “The Murine Version of BAN2401(mAb158) Selectively Reduces Amyloid-Beta Protofibrils in Brain andCerebrospinal Fluid of tg-ArcSwe Mice” J Alzheimers Dis 43:575-588(2015), Lambert et al., “Monoclonal Antibodies that Target PathologicalAssemblies of Abeta” J Neurochem 100:23-35 (2007), Hillen et al.,“Generation and Therapeutic Efficiency of Highly Oligomer-SpecificBeta-Amyloid Antibodies” J Neurosci 30:10369-10379 (2010), Rasool etal., “Systemic Vaccination with Anti-Oligomeric Monoclonal AntibodiesImproves Cognitive Function by Reducing Abeta Deposition and tauPathology in 3×Tg-AD mice” J. Neurochem 126:473-482 (2013), Dorostkar etal., “Immunotherapy Alleviates Amyloid-Associated Synaptic Pathology inan Alzheimer's Disease Mouse Model” Brain 137:3319-3326 (2014),Castillo-Carranza et al., “Tau Immunotherapy Modulates Both PathologicalTau and Upstream Amyloid Pathology in an Alzheimer's Disease MouseModel” J Neurosci 35:4857-4868 (2015), and O'Nuallain et al.,“Conformational Abs Recognizing a Generic Amyloid Fibril Epitope” ProcNatl Acad Sci USA 99:1485-1490 (2002), which are each herebyincorporated by reference in their entirety). However, none of thesemAbs are specific for secondary structure or were raised to anon-self-antigen, and none have been shown to bind both Aβ oligomers, aswell as pathological tau (PHF). All is a rabbit polyclonal antibodyraised to Aβ1-40 bound to gold colloid particles (Kayed et al.,“Conformation-Dependent Anti-Amyloid Oligomer Antibodies” MethodsEnzymol 413:326-344 (2006), and Kayed et al., “Common Structure ofSoluble Amyloid Oligomers Implies Common Mechanisms of Pathogenesis”Science 300:486-489 (2003), which are both hereby incorporated byreference in their entirety). NAB61 was generated using Aβ1-40crosslinked with peroxynitrile (Lee et al., “Targeting Amyloid-BetaPeptide (Abeta) Oligomers by Passive Immunization with aConformation-Selective Monoclonal Antibody Improves Learning and Memoryin Abeta precursor Protein (APP) Transgenic Mice” J Biol. Chem281:4292-4299 (2006), which is hereby incorporated by reference in itsentirety). BAN2401 was raised to protofibrils of Aβ1-42 with the Arcticmutation (Tucker at al., “The Murine Version of BAN2401 (mAb158)Selectively Reduce Amyloid-Beta Protofibrils in Brain and CerebrospinalFluid of tg-ArcSwe Mice” J Alzheimers Dis 43:575-588 (2015), which ishereby incorporated by reference in its entirety). NU-1 was raised toamyloid β-derived diffusible ligands (ADDLs) of Aβ1-42 (Lambert et at,“Monoclonal Antibodies That Target Pathological Assemblies of Abeta” JNeurochem 100:23-35 (2007), which is hereby incorporated by reference inits entirety). A-887755 was raised to globulomers of Aβ20-42 (Hillen atal., “Generation and Therepeutic Efficacy of Highly Oligomer-SpecificBeta-Amyloid Antibodies” J Neurosci 30: 10369-10379 (2010), andDorostkar et al., “Immunotherapy Alleviates Amyloid-Associated SynapaticPathology in an Alzheimer's Disease Mouse Model” Brain 137:3319-3326(2014), which are both hereby incorporated by reference in theirentirety). ACU-193 was raised to aggregated Aβ (Goure et al., “Targetingthe Proper Amyloid-Beta Neuronal Toxins: a Path Forward for Alzheimer'sDisease Immunotherapeutics” Alzheimers Res Ther 6:42 (2014), which ishereby incorporated by reference in its entirety). B10 is an antibodybinding domain selected for stabilizing Aβ protofibrils (Habicht et al.,“Directed Selection of a Conformational Antibody Domain that PreventsMature Amyloid Fibril Formation by Stabilizing Abeta Protofibrils” ProcNatl Acad Sci USA 104:19232-19237 (2007), which is hereby incorporatedby reference in its entirety). The two IgMs W01 and W02, both raisedagainst Aβ, only recognize generic amyloid fibrils and protofibrillar Aβbut not soluble oligomeric forms (O'Nuallain et al., “Conformational AbsRecognizing a Generic Amyloid Fibril Epitope” Proc Natl Acad Sci USA99:1485-14900 (2002), which is hereby incorporated by reference in itsentirety). The same group, that produced All, has also produced OC, apolyclonal, which was raised to Aβ1-42 fibrils (Kayed et al., “FibrilSpecific, Conformation Dependent Antibodies Recognize a Generic EpitopeCommon to Amyloid Fibrils and Fibrillar Oligomers That is Absent inPrefibrillar Oligomers” Mol. Neurodegener 2:18 (2007), which is herebyincorporated by reference in its entirety) and developed the rabbit IgGmAbs 204 and 205 generated using Aβ1-40 coupled to colloidal goldparticles (Rasool et al., “Systemic Vaccination with Anti-OligomericMonoclonal Antibodies Improves Cognitive Function by Reducing AbetaDeposition and Tau Pathology in 3×Tg-AD Mice” J Neurochem 126:473-482(2013), which is hereby incorporated by reference in its entirety). Noneof these aforementioned antibodies directly recognize tau oligomers/taurelated pathology. Furthermore, the majority of these mAbs have beencharacterized with unspecific chemical methods such as dot blots. Hence,although they likely have high affinity for certain specific oligomerspecies, they might also bind to appropriately folded monomers at alower affinity. In potential therapeutic settings the concentration ofphysiological monomeric species is much higher, hampering theeffectiveness of such antibodies. Furthermore, all of these mAbs wereraised and selected by variations of the same Aβ self-antigen, havingthe potential issue of late autoimmune toxicity. On the other hand, theanti-tau oligomer specific mAbs (TOMA) have also been raised, using theaggregated tau self-antigen, and shown to reduce tau pathology(Castillo-Carranza et al., “Tau Immunotherapy Modulates BothPathological Tau and Upstream Amyloid Pathology in an Alzheimer'sDisease Mouse Model” J Neurosci 35:4857-4868 (2015), which is herebyincorporated by reference in its entirety); however, these do notcross-immunoreact with Aβ oligomers.

Due to the novel method by which the anti-β-sheet conformational mAbswere generated and their poly-reactivity to toxic conformers found inmost common NDD, the approach described herein is innovative and morelikely to have therapeutic success in humans than any of the otherexisting oligomer targeting mAbs. The potential advantages of the anti-βsheet mAbs described herein are: 1) a diminished risk of inducingauto-immune complications since the immunogen used has no sequencehomology to any human peptide/protein (except to the protein expressedin the very rare patients with British amyloidosis); 2) selectivetargeting of the β-sheet secondary structure found in toxic oligomers,thus avoiding interference with the multiple physiological functions ofsoluble Aβ, tau and α-synuclein; 3) reduced risk of inducing vasogenicedema/encephalitis related to direct clearance of fibrillar Aβ vasculardeposits, since mainly oligomeric forms of Aβ and tau are beingtargeted; 4) concurrently targeting Aβ, tau and α-syn related pathologicconformers, addressing the mixed pathologies found in the majority ofNDD patients (Hamilton et al., “Lewy Bodies in Alzheimer's Disease: ANeuropathological Review of 145 Cases Using Alpha-SynucleinImmunohistochemistry” Brain Pathol 10:378-384 (2000), White et al.,“Neuropathologic Comorbidity and Cognitive Impairment in the Nun andHonolulu-Asia Aging Studies” Neurology 86:1000-1008 (2016), Schneider etal., “Mixed Brian Pathologies Account for Most Dementia Cases inCommunity-Dwelling Older Persons” Neurology 69:2197-2204 (2007), andJames et al., “TDP-43 Stage, Mixed Pathologies, and ClinicalAlzheimer's-Type Dementia” Brain (2016), each of which is herebyincorporated by reference in its entirety); 5) minimal risk ofincreasing toxic oligomer species as shown in some vaccination methods(Hara et al., “An Oral Abeta Vaccine Using a RecombinantAdeno-Associated Virus Vector in Aged Monkeys: Reduction in PlaqueAmyloid and increase in Abeta Oligomers” J Alzheimers Dis 54:1047-1059(2016), which is hereby incorporated by reference in its entirety); 6)the possible use in prion diseases with the potential to interfere withthe spread of PrP^(Res) No other reported methodologies to produce mAbsto oligomers published thus far have this unique combination ofproperties. Hence, the technological approach described herein has thepotential to develop tools for the detection, monitoring and treatmentof multiple NDDs.

Example 2—Characterization of Anti-β-Sheet Conformational mAb aβComAbWG-3D7

The purification of the anti-β-sheet conformational monoclonal antibodyaβComAb WG-3D7 is depicted in FIGS. 11A-11B. FIG. 11A shows westernblots of the conformational monoclonal antibody WG-3D7 purified with allama anti-μ column. The far left panel shows Fast Green staining forprotein loading control, the middle panel shows anti-mouse IgM μspecific reactivity, and the right panel shows anti-mouse Kappareactivity. Lanes 1 and 2 of each panel contain un-reduced and DTTreduced proteins, respectively (IgMk p=pentamer, Hμ r=reduced heavychain, and Kr=Kappa light chain reduced). FIG. 11B contains graphs ofELISA data showing the reactivity of the purified conformationalmonoclonal antibody WG-3D7 against (in order from left to right on thegraphs) Aβ40, oligomerized Aβ42, and PHF (left graph), and positive andnegative controls showing that the antigen specificity of the WG-3D7antibody (solid bars) is not an artifact of the secondary anti-IgM(hatched bars) (right graph).

The reactivity of the anti-β-sheet conformational monoclonal antibodyaβComAb WG-3D7 against aggregated/oligomeric amyloid β and PHF is shownby electron microscopy in FIGS. 12A-12E FIGS. 12A and 12B showfibrilized and polymerized Aβ1-42, respectively. FIGS. 12C and 12D showPHF and Protein Kinase A (PKa) digested PHF (arrow fibril; arrowheadoligomer). FIG. 12E, middle panel, is a Western blot showing thereactivity of the conformational antibody WG-3D7 against PHF and PKadigested PHF (lanes 1 and 2), and Aβ42 fibrilized and polymerized forms(lanes 3 and 4). FIG. 12E, right panel, is a Western blot showingreactivity of PHF-1 IgG against PHF and PKa digested PHF (lanes 1 and2), and reactivity of 468/6E10 IgG against AB42 fibrilized andpolymerized forms (lanes 3 and 4). FIG. 12E, left panel shows the sameblot as in middle and right panels, but stained with Fast Green to showprotein loading.

Binding of aβComAb WG-3D7 to oligomeric Aβ42 is shown in FIGS. 13A-13B.Surface plasmon resonance indicates binding affinity of theconformational antibody WG-3D7 to the oligomeric species of Aβ42 (FIG.13B, left graph). In contrast there is no detectable binding to themonomeric form of Aβ42 (FIG. 13B, right graph). The normalized data ofbinding oligomer Aβ42 (FIG. 13A) was used to determine the associationand dissociation rate constants also shown in FIG. 13A (right-handtable).

Immunohistochemical reactivity of aβComAb WG-3D7 on AD, age-matched, andyoung control human brains sections is shown in FIGS. 14A-14B. FIG. 14Ashows representative images of WG-3D7 immunoreactivity in the entorhinalcortex of AD (left image), age-matched control (middle image), and youngcontrol (right image) brain sections. FIG. 14B are representative imagesshowing WG-3D7 immunoreactivity in AD vulnerable brain regions (i.e.,CA4, CA3, CA1, subiculum and entorhinal cortex) (top panel of images)versus control brain (bottom panel of images). For bothimmunofluorescent and DAB immunohistochemistry experiments, brains werestained in parallel in one experimental run. Brain slides were alsotreated and imaged at the same settings. Scale bar indicates 100 μm.

Immunohistochemistry of anti-β-sheet conformational monoclonal antibodyaβComAb WG-3D7 on human AD brains sections is depicted in FIGS. 15A-15B.FIG. 15A shows representative images of co-staining of intraneuronal(arrowhead) and amyloid plaques (arrows) with commercial anti-Aβspecific 4G8/6E10 antibodies (black stain) and conformational antibodyWG-3D7 (red-gray stain) in AD human brain tissue. FIG. 15B showsimmunofluorescence of conformational monoclonal antibody WG-3D7 on humanAD brain tissue alone (far left image), localization of Aβ using4G8/6E10 antibody (FIG. 15B, top middle image), and co-localization ofWG-3D7 and 4G8/6E10 staining (far right image). Lower middle image ofFIG. 15B shows magnification of the area boxed in the co-localizationimage (i.e., FIG. 15B, right image). Scale bar indicates 50 μm. Whitearrows show intracellular co-staining of WG-3D7 antibody and amyloid β,grey arrows show extracellular material and plaques co-labeling withWG-3D7 and 4G8/6E10. Red staining appears to indicate WG-3D7 staining ofoligomeric forms of Aβ within the plaques and the surroundings.

The co-localization of aβComAb WG-3D7 and pathological Tau species inhuman AD brain sections is demonstrated in FIGS. 16A-16B. FIG. 16A showsimmunofluorescence of conformational monoclonal antibody WG-3D7 (leftimage) and PHF-1 IgG (middle image) on human AD brain tissue.Co-localization of WG-3D7 and PHF-1 antibody (in white) staining isshown in FIG. 16A (right side image). FIG. 16B shows immunofluorescenceof the conformational monoclonal antibody WG-3D7 (left side image) andantibody AT8 (middle image). Co-localization of WG-3D7 and AT8 staining(in white) is shown in FIG. 16B, right side image. Scale bars indicate50 μm.

Example 3—Characterization of Anti-β-Sheet Conformational mAb aβComAbFT-11F2

The purification of the anti-β-sheet conformational monoclonal antibodyFT-11F2 with SAS and quantification of the antibody binding levels topathological conformers is depicted in FIGS. 17A-17B. FIG. 17A showsWestern blots of the conformational monoclonal antibody FT-11F2, whichwas initially purified using 50% SAS. The left panel shows Fast Greenstaining for protein loading, the middle panel shows anti-mouse IgM μspecific reactivity, and the right panel shows anti-mouse Kappa specificreactivity. Lane 1 contains un-reduced sample (IgMk p=pentamer) and lane2 contains 0.1 M dithiothreitol (DTT) reduced sample (Hμ, r=reducedheavy chain and Kr=Kappa light chain reduced). FIG. 17B are graphs ofELISA data showing the reactivity of the purified conformationalmonoclonal antibody FT-11F2 against (in order from left to right on thegraph) Aβ40, oligomerized Aβ42, PHF, and PrP (left graph). The rightgraph of FIG. 17B shows plate coating antigen controls. Immobilizedantigen (i.e., Aβ40, oligomerized Aβ42, PHF, and PrP, in order from leftto right on the graph) were bound by primary antibodies anti-Aβ 4G8/6E10(IgG) antibody, PHF-1 (IgG) antibody, and anti-PrP 7D9/6D11 (IgG)antibody, and detected by a secondary antibody anti-mouse IgG. Thisreaction served as a positive control of the even distribution of theantigens seeded on the plates. Incubation with the same primaryantibodies (solid bars) and secondary anti-IgM (hatched bars) served asa control for possible non-specific binding of the secondary anti-IgM.This data corroborates the specific binding of the IgM monoclonals tothe respective antigens in the left graph.

The reactivity of anti-β-sheet conformational mAb FT-11F2 againstnormal, fibrilized, and oligomeric α-synuclein and fibrils of purifiedhuman PHF is shown in FIGS. 18A-18B. FIG. 18A shows electron microscopyimages of normal α-synuclein (1), fibrilized α-synuclein (2) andoligomeric forms of α-synuclein after polymerization with glutaraldehyde(3-3″′). There are different oligomeric states where size andaggregation varies, from small individual oligomers (3), graduallyincreasing its size (3′ and 3″) and finally bigger oligomer aggregates(3″′). Far right panels of FIG. 18A show PHF (4) and PKa digested PHF(5). FIG. 18B shows Western blots of the reactivity of theconformational antibody FT-11F2 (second blot) against normal α-synuclein(lane 1), fibrilized α-synuclein (lane 2), and oligomeric α-synuclein(lane 3) at different states (as shown in the EM images), and PHF (lane4). The third and fourth blots of FIG. 18B show reactivity of antiα-synuclein and PHF-1 specific antibodies, respectively, which alsocross-react with fibrilized and oligomeric α-synuclein. Arrows depictdifferent oligomers of α-synuclein (as shown in FIG. 18A) that arerecognized also by the FT-11F2 clone (lane 3).

The immunoreactivity of anti-β-sheet conformational mAb aβComAb FT-11F2against oligomeric α-synuclein forms surrounding Lewy bodies inParkinson's disease in human brains is shown in FIG. 19. Representativeimages showing immunoreactivity of the conformational monoclonalantibody FT-11F2 (second panel of images) and anti α-synuclein antibody(third panel of images) on human brain sections affected by Parkinson'sdisease in the substantia nigra. As shown in the fourth panel of images,conformational antibody FT-11F2 does not co-localize directly with thefibrillar α-synuclein that is found within Lewy bodies, but in thecytoplasm of substantia nigra neurons in the vicinity of the Lewybodies. FT-11F2 does not immunolabel substantia nigra neurons fromcontrol, non-Parkinson's disease patients.

Example 4—Characterization of Anti-β-Sheet Conformational mAb aβComAbFT-12E1

aβComAb FT-12E1 reactivity against Aβ 1-40, oligomerized Aβ 1-42, humanpurified PHF, and oligomerized, recombinant deer Prion protein (dPrP) isshown in FIG. 20. The right graph of FIG. 20 shows plate coating antigencontrol. Incubation of the immobilized antigen (i.e., from left toright, Aβ40, oligomerized Aβ42, PHF, and oligomerized dPrP) with primaryantibodies (i.e., anti-Aβ 4G8/6E10 (IgG) antibody, PHF-1 (IgG) antibody,and anti-PrP 7D9/6D11 (IgG) antibody) and a secondary anti-mouse IgGantibody, served as a positive control for the even distribution of theantigens seeded on the plates. Incubation with the same primaryantibodies (solid bars) and secondary anti-IgM (hatched bars) (rightgraph) served as a control for possible non-specific binding of thesecondary anti-IgM. This data corroborates the specific binding of IgMmonoclonals to the respective antigens in the left histogram.

Purification of the anti-β-sheet conformational monoclonal antibodyFT-12E1 is depicted in FIGS. 21A-21B. FIGS. 21A and 21B are westernblots of the conformational monoclonal antibody FT-12E1 purified with allama anti-μ column. FIG. 21A shows Fast Green staining as a proteinloading control. FIG. 21B shows anti-mouse IgM μ specific (left panel)and anti-mouse Kappa reactivity (right panel). Lane 1 of each panelcontains un-reduced sample and lane 2 contains DTT reduced protein (IgMkp=pentamer, Hμ r=reduced heavy chain, and Kr=Kappa light chain reduced).

The reactivity of anti-β-sheet conformational mAb aβComAb FT-12E1against purified human PHF, three different strains of pathogenicPrP^(Res) and α-synuclein is shown in FIGS. 22A-22C. FIG. 22A showselectron microscopy images of PHF (1) and PKa digested PHF (2) (greyarrow fibril; black arrowhead oligomer), normal α-synuclein (3),fibrilized α-synuclein (4), and oligomeric forms of α-synuclein afterpolymerization with glutaraldehyde (5-5″′). Different oligomeric statesof α-synuclein having a variety of sizes and aggregation states aredepicted, from small single oligomers (5), to those incrementallyincreasing its size (5′ and 5″), and finally bigger oligomer clusters(5″′). FIG. 22B (middle panel) is a Western blot showing the reactivityof the conformational monoclonal antibody FT-12E1 against PHF and PKadigested PHF (lanes 1 and 2), two different prion scrapie strains 22L,139A and recombinant deer PrP (lanes 3, 4 and 5 respectively). Theright-hand blot shows reactivity of PHF-1 IgG, and PrP specificantibodies 7D9/6D11 against the aforementioned protein samples aspositive controls. The left-hand blot of FIG. 22B shows Fast Greenstaining of the middle and right blots as a protein loading control.FIG. 22C shows a series of four Western blots. The second blot in theseries shows reactivity of the conformational antibody FT-12E1 againstnormal (lane 1), fibrilized (lane 2), and oligomeric (lane 3)α-synuclein, where FT-12E1 recognizes α-synuclein oligomers. The thirdand fourth blots of FIG. 22C show anti α-synuclein IgG and PHF-1 IgGreactivity against fibrilized and oligomeric α-synuclein samples loadedin the same lanes as described above. The first blot of FIG. 22C is aFast Green protein loading control.

The reactivity of anti-β-sheet conformational mAb FT-12E1 in AD humanbrain sections is depicted in FIGS. 23A-23B and FIGS. 24A-24C. FIG. 23Ashows immunoreactivity of the conformational monoclonal antibody FT-12E1is preferential to glial cells (black arrows) rather than neurons (blackarrowhead). FIG. 23B (control) shows there is no staining in non-AD,control brain sections. The right images of FIGS. 23A and 23B aremagnifications of the boxed areas shown in the left images of eachfigure. 24A is a representative image showing the staining pattern ofthe conformational monoclonal antibody FT-12E1 in a human AD brainsection. FIGS. 24B and 24C are magnified views of the boxed areas,showing characteristic cytoplasmic staining in glial-like cells andalong processes.

Example 5—Characterization of Anti-β-Sheet Conformational mAbs aβComAbsTF-10E8 and TF10F7

Purification of the anti-β-sheet conformational monoclonal antibodiesTF-10E8 and TF-10F7 with a llama anti-μ column is depicted in FIGS.25A-25B. FIG. 25A shows Fast green staining as a protein loadingcontrol. FIG. 25B shows anti-mouse IgM μ specific reactivity (left blot)and anti-mouse Kappa reactivity (right blot). Lanes 1 and 2 of eachpanel contain un-reduced TF-10E8 and TF-10F7 antibodies, respectively,and lanes 3 and 4 of each panel contain DTT reduced TF-10E8 and TF-10F7antibodies, respectively. IgMk p=pentamer, Hμ r=reduced heavy chain, andKr=Kappa light chain reduced.

The reactivity of mAbs TF-10E8 and TF-10F7 against PHF and threedifferent strains of pathogenic PrP is demonstrated in FIGS. 26A-26C.FIG. 26A shows electron microscopy images of PHF (1) and PHF digestedwith PKa (2) (grey arrow fibril; black arrowhead oligomer). Typicaloligomerized PrP from single oligomers (3) that start aggregating (4 and5) to form bigger clusters of oligomers (6). FIGS. 26B and 26C areWestern blots showing the reactivity of the conformational monoclonalantibodies TF-10E8 (FIG. 26B, middle) and TF-10F7 (FIG. 26C, middle)against PHF (lane 1) PKa digested PHF (lane 2), prion scrapie strains22L (lane 3) and 139A (lane 4), and recombinant deer PrP (lane 5).Protein loading of the aforementioned blots was visualized using FastGreen staining (left most blots shown in FIGS. 26B and 26C). Positivecontrol staining was carried out using PHF-1 IgG and 7D9/6D11 (Prp) IgGas shown in the right-most blots of FIGS. 26B and 26C.

The immunoreactivity of anti-β-sheet conformational mAb TF-10E8 in ADaffected human brain hippocampus tissue is shown in FIGS. 27A-27B. FIG.27A shows representative images of the specificity of the conformationalmonoclonal antibody TF-10E8 for neuronal cytoplasm (black arrowhead),neuronal processes (black arrows) and with less intensity nuclei (whitearrows) of neurons. FIG. 27B shows no TF-10E8 immunoreactivity inage-matched control brain sections. The right-hand images of FIGS. 27Aand 27B, respectively, are higher magnification views of the boxed areasidentified in left-hand images of each respective figure.

The immunoreactivity of anti-β-sheet conformational mAb TF-10F7 in ADaffected human brain sections is shown in FIGS. 28A-28B. FIG. 28A showsrepresentative images of the specificity of the conformationalmonoclonal antibody TF-10F7 for neuronal cytoplasm (black arrowhead),neuronal processes (black arrows), and nuclei (white arrows) of neurons.Some stained neurons are in close proximity to each other (consistentwith the hypothesis that pathological oligomeric conformers can spreadfrom neuron to neuron). It is possible to detect dystrophic neurons(white arrowheads). FIG. 28B are images showing no TF-10F7immunoreactivity in age-matched control brain sections. The right-handimages of FIGS. 28A and 28B, respectively, are higher magnificationviews of the boxed areas identified in the left-hand images of eachrespective Figure.

The immunoreactivity of anti-β-sheet conformational mAbs aβComAbsTF-10E8 and TF-10F7 in human brain sections of a subject havingGerstmann-Straussler-Scheinker syndrome (GSS), a genetic, autosomaldominant prion disease is shown in FIGS. 29A-29B. FIGS. 29A and 29B arerepresentative images showing specificity of the conformationalmonoclonal antibodies TF-10E8 and TF-10F7 for neuronal cytoplasm,neuronal processes, and the nucleus of neurons in the GSS human brainsections (arrows). The right-hand images of FIGS. 29A and 29B showmagnified views of the boxed areas identified in the left-hand images.

Example 6—Anti-β-Sheet Conformation Monoclonal Ameliorates Aβ and TauOligomer Pathology in an Alzheimer's Mode

Production and Selection of the anti β-sheet Conformational MonoclonalAntibody GW-23B7. Anti-β-sheet conformational monoclonal antibody(aβComAb) GW-23B7 was obtained after immunization of a CD-1 mouse withthe p13Bri immunogen and subsequent hybridoma production performed atthe Bi-Institutional Antibody and BioResource Core Facility of MemorialSloan Kettering Cancer Center and The Rockefeller University asdescribed in Example 1. All procedures were approved by theInstitutional Animal Care and Use Committee protocol #97-03-009 andfollowed NIH standards.

Selection of a conformational monoclonal with reactivity to oligomerspresenting dominant β-sheet secondary structure was done byenzyme-linked immunosorbent analysis (ELISA) as previously described(Goñi et al., “Immunomodulation Targeting of Both Abeta and TauPathological Conformeres Ameliorates Alzheimer's Disease Pathology inTgSwDl and 3×Tg Mouse Models” J Neuroinflammation 10:150 (2013), andGoñi et al., “Immunomodulation Targeting Abnormal Protein ConformationReduces Pathology in a Mouse Model of Alzheimer's Disease” PLoS. ONE5(10):e13391 (2010), which are both hereby incorporated by reference intheir entirety). Plates were coated overnight at 4° C. with eitherAβ1-40 or Aβ1-42 in 50 mM ammonium sulfate pH 9.6 maximizing theoligomeric content difference between both Aβ peptides (FIGS. 30A-30E),paired helical filaments (PHF) purified from a human brain ofAlzheimer's disease (AD) or PrP^(res) as previously described in Example1 (Goñi et al., Immunomodulation Targeting Abnormal Protein ConformationReduces Pathology in a Mouse Model of Alzheimer's Disease” PLoS ONE5(10):e13391 (2010), which is hereby incorporated by reference in itsentirety. Approximately 125 μL of cell supernatant was diluted 1:1 with50 mM Tris-buffered saline pH 7.2, 0.1% Tween 20 (TBS-T) and 50 μl/wellwere applied to each one of the four Immulon 2HB (Thermo, Waltham,Mass.) 96-well microtiter pre-coated plates. Bound antibodies weredetected in the original cloning with horseradish peroxidase-labeled(HRP) goat anti-mouse IgG+IgM+IgA (H+L) (KPL, Gaithersburg, Md., USA).Color developing substrate was Tetramethyl benzidine (TMB; Pierce,Rockford, Ill.) and readings were made at 450 nm. aβComAb GW-23B7 wasfurther sub-cloned and levels of reactivity tested as above induplicates diluted 1:1000 in TBS-T and detected with HRP-goat anti-mouseIgM(μ) (KPL, Gaithersburg, Md., USA) or HRP-goat anti-mouse Kappa chain(Southern Biotech, US). Control coating was assessed with commercial4G8/6E10 antibodies specific for Aβ peptides, PHF-1 for PHF and 7D9/6D11antibodies for PrP; all detected with HRP-goat anti-mouse IgG (H+L) (GEHealthcare UK). Duplicates of the controls were used to determine thelack of cross-reactivity of the secondary anti-μ with the IgGs or thecoating protein/peptides

Purification of aβComAb GW-23B7. The aβComAb GW-23B7 present in thesubcloned hybridomas was precipitated with Saturated Ammonium Sulfate(SAS) 761.5 g/Lt at 21° C. Samples were made 30% in SAS, incubated atRoom temperature (RT) for at least 4 hours, centrifuged at 14,000×g for15 min and the precipitate washed with a comparable volume of 30% SASand stored at 4° C. until further use.

Partially purified aβComAb GW-23B7 IgM was further purified throughCaptureSelect (Thermo-Life Technologies, Waltham, Mass.) IgM affinitymatrix containing a 14 kDa Llama antibody fragment recognizingspecifically human or mouse IgM but none other immunoglobulins (IgG,IgA) from any animal. The ligand coupled to NHS-activated Sepharose 4Fast Flow has a binding capacity of 2.5 mg of IgM per ml of matrix.Briefly, 1 ml of matrix in 20% ethanol was poured at RT into a 3 mlplastic column, equilibrated and washed with at least 20 bed volumes ofPBS pH 7.2. The column was drained to the top before adding the samplediluted in PBS. The flow rate was established at 1 ml/min and thecollected flow was passed again at least three times through the columnto maximize binding. The column was then washed with 5 bed volumes ofPBS before eluting the IgM with 1.5 bed volumes of 0.1 M Glycine pH 3.0;the eluates were immediately neutralized with 1N NaOH. A second 1.5 bedvolume of 0.1M glycine pH 3.0 was added to release all the bound IgM andthe column was regenerated with 10 bed volumes of PBS to start theprocess again as needed. The eluted IgM was aliquoted and kept at −80°C. until use. Each batch of IgM aβComAb GW-23B7 was assessed for purityon blots with specific antisera and protein stain, and for antibodyactivity against oligomers run on gels or ELISA as described above.

Surface Plasmon Resonance. Measurements of the affinity of aβComAbGW-23B7 to Aβ oligomeric conformers were determined using surfaceplasmon resonance. Briefly, a carboxymethyl dextran gold sensor slidewas activated with a 10 mg/ml N-hydroxysuccinimide/40 mg/mlN-ethyl-N′-(dimethylaminopropyl) carbodiimide aqueous solution injectedover both channels of a Reichert SR 7000DC surface plasmon resonancesystem (Reichert Technologies, Depew, N.Y.) for 7 minutes at 20 uL/min.Then a 50 ug/mL solution of either glutaraldehyde polymerized Aβ1-42 orHFIP monomeric Aβ1-42 in 10 mM sodium acetate pH 5.0 buffer was injectedover only one channel for 20 minutes at 20 μL/min, followed by blockageof excess activated groups by injection over both channels of 1Methanolamine HCl pH 8.5 for 10 minutes. Kinetic measurements wereperformed at 25° C. with a flow rate of 25 μL/min using serial dilutionsof the monoclonal antibody in PBS-T buffer pH 7.4. After each bindingcycle the surface was regenerated by a short and fast injection of 10 mMhydrochloric acid. The plots and the KD were determined using theadvanced biosensor data processing and analyzing Scrubber software 2.0a(Biologic Software).

Electron Microscopy. Samples of Aβ1-40 and Aβ1-42 dissolved in ammoniumbicarbonate pH 9.6 as used for ELISA assay coating, or samples offibrilized and polymerized Aβ 1-42, Paired Helical Filament (PHF) andProtein Kinase A digested PHF diluted 1 mg/ml in PBS pH 7.4 were allapplied, 3 μl of each, onto carbon coated 400 mesh Cu/Rh grids (TedPella Inc., Redding, Calif.) and stained with 1% uranyl acetate indistilled water (Polysciences, Inc, Warrington, Pa.). Stained grids wereexamined under Philips CM-12 electron microscope and photographed with aGatan (4 k×2.7 k) digital camera.

Infusion of 3×Tg mice with aβComAb GW-23B7 or Vehicle Control. Aschematic of the protocol for infusion is shown in FIG. 35A. Allprocedures were approved by the NYU Institutional Animal Care and UseCommittee protocol #170202-01 and followed NIH standards. Two groups(n=10) of 16 month old triple transgenic Alzheimer's Disease Mouse modelAPP KM670/671NL (Swedish), MAPT P301L, PSEN1 M146V (Oddo et al.,“Triple-Transgenic Model of Alzheimer's Disease with Plaques andTangles: Intracellular Abeta and Synaptic Dysfunction” Neuron39(3):409-21 (2003), which is hereby incorporated by reference in itsentirety) (3×Tg) with amyloid and tau pathologies, received a weeklyintraperitoneal injection of either 100 μL of 1 μg/μL aβComAb GW-23B7 insterile saline or 100 μL of sterile saline for the initial two weeks.Animals were rested for one week and received the same type of weeklyinjections for the following 3 weeks. After two weeks rest, both groupswere inoculated weekly with either 150 μL of 1 μg/μL GW-23B7 or 150 μLof sterile saline control for two more weeks. One week after the lastinjection, locomotor and behavioral cognitive tests were performed forboth groups spanning for three weeks of tests after which the animalswere bled from the heart, thoroughly perfused with PBS pH 7.2 andsacrificed. The brains were harvested immediately, the cerebellum wasremoved, and the remainder split in half, one half flash frozen over dryice for future biochemical studies and the other half fixed inperiodate-lysine-paraformaldehyde (PLP) for histochemistry.Intraperitoneal cavity was visually checked for anomalies and kidneys,spleen and liver were collected for eventual controls.

Two additional groups of 18 m.o. 3×Tg mice (n=12 each group) were usedto assess the kinetics of the IgM monoclonal penetration in the brainafter a peripheral injection. The animals in each group received anintraperitoneal injection of either 150 μL of 1 μg/μL of mAb GW-23B7 insterile saline or 150 μL sterile saline vehicle alone. Four animals ofeach group were euthanized at 6, 24 and 48 hours; extensively perfusedwith PBS (Goñi et al., “Immunomodulation Targeting of Both Abeta and TauPathological Conformers Ameliorates Alzheimer's Disease Pathology inTgSwDl and 3×Tg Mouse Models” J. Neuroinflammation 10:150 (2013), andScholtzova et al., “Amyloid β and Tau Alzheimer's Disease RelatedPathology is Reduced by Toll-like Receptor 9 Stimulation” ActaNeuropathol. Comm. 2:101 (2014), which are both hereby incorporated byreference in their entirety) and the brains harvested for biochemicaland histochemical assays as above described.

Locomotor and Behavioral testing. Locomotor and behavioral cognitivetests were performed as previously described (Goñi et al.,“Immunomodulation Targeting of Both Abeta and Tau PathologicalConformers Ameliorates Alzheimer's Disease Pathology in TgSwDl and 3×TgMouse Models” J. Neuroinflammation 10:150 (2013), and Scholtzova et al.,“Amyloid β and Tau Alzheimer's Disease Related Pathology is Reduced byToll-like Receptor 9 Stimulation” Acta Neuropathol. Comm. 2:101 (2014),which are both hereby incorporated by reference in their entirety).Prior to each test mice were adapted for 15 min to the room lights.Locomotor tasks were performed to verify that any treatment-relatedeffects were not explained by sensorimotor abilities.

Traverse beam. Traverse beam test was used to determine general motorcoordination and balance. Each mouse was placed on a beam 1.1 cm wideand 50.8 cm long supported 30 cm above a padded surface, with a blackbox attached to the end of the beam. Mice were monitored for a maximumtime of 60 sec, recording the number of footslips before falling orreaching the goal box. When an animal fell, it was placed back at theposition it was prior to the fall. The average number of footslips asper four successive trials was calculated. A footslip was defined as anerror and recorded numerically.

Rotarod. Each animal was placed on a Rotarod 7650 accelerating modelapparatus with a diameter of 3.6 cm (Ugo Basile, Biological ResearchApparatus, Varese, Italy) to assess differences in balance and forelimband hindlimb motor coordination. The animals were adapted to theapparatus for two training sessions and tested three times withincreasing speed. The rotor rod was initially set at 1 rpm and speedgradually increased every 30 sec. Latency to fall or invert from the topof the rotating barrel was recorded. The rod was cleaned with water and30% ethanol after each session.

Radial arm maze. Spatial learning was evaluated using an 8 arm radialarm maze (arms 35 cm length and 7 cm wide) with a cup of 1 cm diameterat the end of each arm. The central octagonal area had 14 cm diameterwith Plexiglas guillotine doors operated remotely by a pulley system.Each cup at the end of the arms was baited with saccharine-flavoredwater. The behavioral test consisted of 5 adaptation days followed by 10trial days. All the animals subjected to the test were deprived of water(only given access to water 2 hours per day). Trial time was set to amaximum of 15 minutes for each animal and the time the animals spent tovisit all arms as well as the number of errors, defined as entries topreviously visited arms, were recorded.

Brain homogenization. Flash frozen brain hemispheres from each animalwere weighted and made 20% w/v on tissue homogenization buffer (THB)containing 20 mM Tris pH 7.4, 250 mM sucrose, 1 mMEthylenediaminetetraacetic acid (EDTA), 2.5 mM Ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N-tetraacetic acid (EGTA) filteredthrough 0.2 μm mesh. Before use, freshly made 1.46 nM pepstatin, 1 mMphenylmethane-sulfonyl-fluoride (PMSF), 1 mM sodium fluoride (NaF) and0.96 mM sodium orthovanadate were added to obtain the working THB. Eachhalf brain in THB was placed on ice and homogenized using a PRO 200Hand-held homogenizer and a 5 mm×75 mm flat bottom generator probe (ProScientific, Monroe, Conn.) for 3 cycles of 30 seconds each at 30,000rpm, pausing for 30 seconds between each homogenization cycle. Theobtained 20% brain homogenates (BH) were aliquoted (200 μL/each) andstored at −80° C. until use.

Half brains of two additional groups of 3×Tg mice infused with aβComAbGW-23B7 or sterile saline and sacrificed at 6, 24 and 48 hrs werehomogenized as above described. Half of the samples of each group werepooled, aliquoted (200 μL/each) and stored at −80° C. until use.

Electrophoresis and Western blot. For electrophoresis to confirm theidentity of aβComAb GW-23B7, 1 μg of antibody with or withoutdithiothreitol (DTT) 0.1 M was mixed with an equal volume of tricinesample buffer (BioRad, Hercules Calif.), electrophoresed on Bolt™ 4-12%Bis-Tris (Thermo, Waltham, Mass.) polyacrylamide gels and system andtransferred onto nitrocellulose membranes (NC) for 1 hour at 386 mA in0.1% 3-(Cyclohexylamino)-1-propanesulfonic acid (CAPS) (Sigma-Aldrich,St. Louis, Mo.)-10% methanol. Equal protein loading was assessed on themembranes using protein reversible stain Fast Green (FG) FCF 0.1%(Fisher Scientific, Waltham, Mass.) in 25% methanol-10% Acetic acid for1 minute, distained with 25% methanol, transferred to distilled waterand scanned for records on a Canon F916900 scanner (Canon Inc, China).Membranes were then washed several times in TBS-T until all reversiblestain was eliminated, blocked 1 hour at RT with 5% non-fat dry milk inTBS-T pH 8.3, and incubated with HRP-Rat anti-mouse IgM(μ) Heavy chainspecific 1:6000 (Life Technologies, Waltham, Mass.) or HRP goatanti-mouse Kappa 1:6000 (Southern Biotech, Birmingham, Ala.). Boundantibodies were detected with ECL detection system (Pierce, Rockford,Ill.) on autoradiography films (MIDSCI, St Louis, Mo.); all films werescanned for records.

To evaluate the reactivity of aβComAb GW-23B7 against Aβ1-40, Aβ1-42(fibrilized and polymerized) and Paired Helical Filaments (PHF)(Fibrillar and Proteinkinase A digested), 1-2 μg of each sample waselectrophoresed, transfer to NC and blotted as indicated above. aβComAbGW-23B7 diluted 1:1000 in TBS-T was incubated with the blots for 1 hourat RT, and bound immunoglobulin detected with HRP anti-mouse IgM 1:2000.Commercial monoclonal anti-Aβ antibodies 4G8/6E10 (BioLegend, San Diego,Calif.) and anti-abnormally phosphorylated tau monoclonal PHF-1 (whichrecognizes phosphorylated serines at positions 396 and 404) were diluted1:4000 and 1:2000 respectively and used as controls for the identity ofpeptides Aβ1-40, Aβ1-42 and PHF.

To test the presence of IgM in the brain of 3×Tg mice treated withaβComAb GW-23B7 or with control sterile saline alone; pools of four 20%BH corresponding to either 6, 24 or 48 hrs. were treated with SAS asfollows: 165 μL of each pool was mixed with 135 μL of SAS, incubated for30 mins at RT in a tube rotator and tubes centrifuged for 6 mins at14,000×g. Pellets were re-suspended in 100 μL of 45% SAS, vortexedrepeatedly and centrifuged. Washed pellets were re-suspendedsequentially in 75 μL of distilled deionized water and 75 μL of tricinesample buffer, centrifuged for 5 mins at 14,000×g at RT. 4 μL of theclear supernatant was mixed with 6 μL of tricine sample buffer with andwithout DTT 0.1 M and electrophoresed on Bolt™ 4-12% Bis-Trispolyacrylamide gels and system. The western blot was performed asdescribed above. Each sample loaded into the gels represented theconcentrated protein of approximately 1/150 of the whole brain to assurethat minimal representation or differences in IgM immunoglobulin couldbe detected. aβComAb GW-23B7 infused animals were analyzed in pools andindividually.

Quantitation of aggregated/oligomeric A/I and phosphorylated tau on 3×Tgmice. Aggregated/oligomeric Aβ levels were determined using the HumanAggregated Aβ ELISA kit (Invitrogen, Camarillo, Calif.), following themanufacturer's instructions. Briefly, 20% BH were thawed and made 1:4 inthe diluents buffer. Samples were applied to the ELISA plates, incubatedfor 2 hs at RT, followed by extensive washing and incubation for 1 h atRT with biotin conjugated detection antibodies which bind only to theimmobilized aggregated Aβ. After removal of excess antibody,HRP-labelled streptavidin was added. Samples were incubated for 30 min,washed and incubated with TMB substrate for color. Intensity of thecolored product is directly proportional to the concentration ofaggregated/oligomeric Aβ in the sample. The standards produced a linearcurve and the best-fit lines determined by linear regression were usedto calculate aggregated Aβ concentrations in the samples.

For the quantification of total tau and phosphorylated tau (Thr 231) theMeso Scale Discovery (MSD) assay (Rockville, Md.) was used as previouslydescribed (Goñi et al., “Immunomodulation Targeting of Both Abeta andTau Pathological Conformers Ameliorates Alzheimer's Disease Pathology inTgSwDl and 3×Tg Mouse Models” J Neuroinflammation 10:150 (2013), whichis hereby incorporated by reference in its entirety). Solublesupernatants of 20% BH from 3×Tg treated and control mice were diluted1:125 with the provided standard diluent buffer and 100 μl aliquotsseeded in each well on the MULTI-SPOT® 96-well 4-spot plate. The plateswere incubated for 2 hs at RT, washed four times for 25 sec each andincubated for 1 hour with the SULFO-TAG™ anti-total tau antibody. Theplates were then washed four times, covered to block the light, andincubated for 25 minutes with HRP-Streptavidin working solution. Thereaction was stopped with stop solution and the plates read on the MSDsystem at 450 nm. All data were recorded and calculations made using thesoftware provided with the MSD system.

Immunohistochemistry. Immunohistochemistry to assess the reactivity ofaβComAb GW-23B7 was performed on old 3×Tg mice (>19 months old) withextensive amyloid-β and tau pathology; or formalin fixed paraffinembedded human cortex sections of Alzheimer's disease (AD), age-matchedcontrols, young control brains and Gerstmann-Straussler-Scheinker (GSS)brains with prion pathology obtained from the Alzheimer Brain Bank ofthe Alzheimer's Disease Center at NYU. All human tissue related studieswere done with appropriate ethical standards under a protocol approvedby the Institutional Review Board at NYU School of Medicine. Writteninformed consent for research was obtained from the patients or legalguardians. All data and samples were coded and handled according to NIHguidelines to protect patients' identities.

Sections were dewaxed followed by rehydration with successive washes ofxylene (2×5 min), 100% ethanol (2×5 min), 95% ethanol (5 min), 70%ethanol (5 min) and phosphate-buffered solution (PBS) (5 min). Next,slides were washed with 0.3% hydrogen peroxide (2×15 min) to quenchendogenous peroxidases, PBS (3×5 min) and 1 hour blocking at RT with 10%normal goat serum [NGS]-0.2% Triton X-100 in PBS. Slides were incubatedovernight at 4° C. with aβComAb GW-23B7 diluted 1:2000 in 3% NGS-0.2%Triton X-100. Slides were washed three times with PBS and incubated withbiotinylated anti-mouse IgM (μ specific chain) antibody (VectorLaboratories, Burlingame, Calif.) diluted 1:1000 in PBS for 1 hourfollowed by 1 hour incubation of Vectastain® AB solution (VectorLaboratories, Burlingame, Calif.). Slides were developed with3,3-diaminobenzidine tetrahydrochloride with 2.5% nickel ammoniumsulfate (Acros Organics, NJ) diluted in 0.2M sodium acetate (NaAc) pH 6.Reaction was stopped by removal of nickel solution and extensivelyrinsing with 0.2 M NaAc, then stabilized with PBS and mounted on glassslides with Depex® Mounting Media (Electron Microscopy Sciences,Hatfield, Pa.).

For immunofluorescence, paraffin embedded human AD or GSS brain sectionswere dewaxed and rehydrated as above described, then blocked 1 hour atRT with 3% NGS-0.2% Triton X-100. Antibodies to be used forco-localization were diluted in the same tube containing PBS, at thefollowing concentrations: aβComAb GW-23B7 1:8000, anti-Aβ antibodies4G8/6E10 1:3000, PHF-1 1:1500 and anti-GFAP 1:1500. Primary antibodieswere incubated overnight at 4° C. Slides were washed with PBS (3×5 min)and incubated 2 hours with Alexa Fluor® 488-conjugated goat anti-mouseIgM (for GW-23B7) and Alexa Fluor® 647-conjugated goat anti-mouse IgG(for commercial antibodies against proteins present in NDDs) (JacksonImmunoResearch, West Groove, Pa.) both diluted 1:500 in PBS. Slides werewashed (3×5 min) and the coverslip was performed with PermaFluor™Aqueous Mounting Medium (Thermo, Waltham, Mass.).

Black and red staining was performed using DAB for aβComAb GW-23B71:8500 as mentioned above. Slides were then washed with PBS (3×15 min),blocked 10 min at room temperature with 3% NGS-0.2%, incubated overnightwith anti-Aβ antibodies 4G8/6E10 diluted 1:3000, washed with PBS (3×5min), incubated with alkaline phosphatase anti-mouse IgG (Sigma-AldrichSt. Louis, Mo.) for 1 hour and color was developed with Vector RedSubstrate (Vector Laboratories, Burlingame, Calif.). Slides were washedwith PBS and mounted on glass slides as described above.

All mouse brains sections fixed with PLP, 40μ/each, were stained aspreviously reported (Goñi et al., “Immunomodulation Targeting of BothAbeta and Tau Pathological Conformers Ameliorates Alzheimer's DiseasePathology in TgSwDl and 3×Tg Mouse Models” J Neuroinflammation 10:150(2013), which is hereby incorporated by reference in its entirety),incubating overnight with monoclonal antibodies 4G8/6E10 1:3000, PHF-11:1500 or aβComAb GW-23B7 1:3000. Secondary antibodies used werebiotinylated anti-mouse IgG (H+L) or anti-mouse IgM (VectorLaboratories, Burlingame, Calif.) diluted 1:1000 in PBS. Slides weredeveloped with DAB in 2.5% nickel ammonium sulfate and mounted on glassslides with Depex® Mounting Media.

Statistical analysis. Statistical analyses were performed in thesoftware GraphPad Prism v.7. For the behavioral testing, two-way ANOVAwere used and two-tailed t tests for biochemistry assays.

Results. Original clone 23B obtained after the inoculation of a CD-1mouse with β-sheet dominant p13Bri antigen and subsequent hybridomaproduction was assessed by an ELISA assay designed to detect thedifferential concentration of Aβ oligomers between Aβ1-40 and Aβ1-42samples aged in alkaline buffer, and the most oligomer laced form ofpaired helical filaments (PHF) extracted from the brain of a confirmedtypical human AD case (FIGS. 30A and 30B). Both misfolded proteins onlyhave in common the β-sheet secondary structure of the abnormalconformers.

Small availability of cell supernatant from the initially viablehybridoma cells allowed only one determination per sample per antigenand detection of all the main classes of immunoglobulins together withtotal anti-mouse GAM antisera. The selection process was explainedelsewhere and involved the measurement of the differential reactivity toAβ40, Aβ42 and PHF of at least three times over the background O.D.readings compared to an irrelevant hybridoma clone from the same fusion(FIG. 30B). The 23B passed three rounds of selection before beingclassified as GW-23B7 anti-β-sheet conformational monoclonal antibody(aβComAb). The GW-23B7 was later grown in a 5 ml tube and thesupernatant showed two main proteins consistent with the albumin fromthe bovine fetal serum supplement (BSA) and a potential immunoglobulinwith a high molecular weight of around 1,000 kDa (FIG. 30C). The BSA wasremoved by partial purification on SAS precipitation, and the ensuingblot showed a high molecular weight band on top of the gel beforereduction and three bands of approximately 76 kDa, 56 kDa and 27 kDaafter reduction with DTT (FIG. 30D). The high molecular weight bandreacted with both specific anti-mouse μ chain and anti-mouse kappa chainas expected for a pentameric IgMk; a small percentage of a monomericform at around 180 kDa was also seen. The reduced material showed theintact HEM at 76 kDa and a small fraction of truncated Hp. chain at 56kDa not an uncommon find in some IgM preparations. The 27 kDa band wasidentified as the reduced kappa L chain (FIG. 30E).

The sub-clone aβComAb GW-23B7 showed by ELISA test significantcross-reactivity with both the differential Aβ oligomers and theoligomeric/fibrilar PHF; both reactivities were present in the sameintact IgM molecule (FIG. 31A left panel). The control of the antigenscoating the plates was done with commercial mouse IgG antibodies forAβ4G8/6E10, that recognized only the specific primary sequence of Aβ40and Aβ42 and both in the same amount, and the commercial mouse IgGantibody PHF-1 specific for hyperphosphorylated tau that only reacted tothe human PHF coating. Neither one of the bound IgG specific antibodieswas detected by the secondary antibody to μ chain, a necessary controlto discard unspecific cross-recognition of the coating antigens by thelabeled secondary antisera (FIG. 31A right panel).

Further purified through a llama V_(HH) anti-mouse IgM column, theaβComAb GW-23B7 was recovered as >99% pure IgMk pentameric with intactheavy and light chains (FIG. 31B) retaining the same cross-reactivity todifferential Aβ oligomers and PHF in ELISA plates after a L1000 dilutionto the original supernatant (FIG. 31C). To corroborate the GW-23B7binding to oligomeric forms of Aβ42, the purified IgMk was run onsurface plasmon resonance (SPR) with gold sensor slides immobilizingeither oligomeric Aβ42 or the HFIP treated monomeric form. The GW-23B7only showed binding affinity for the oligomeric form with a K_(D) in thenanomolar range (FIG. 31D).

To determine the pathology specificity of the cross-reactivity that byELISA and SPR was shown to be related to the shared β-sheet secondarystructure present in both oligomeric forms of Aβ and ptau, the GW-23B7was used for immunohistochemistry in samples from human brains of eitherknown cases of Alzheimer's disease, age matched brain from patients withno clinical cognitive manifestations, and brains from young adults withno known neuropathology. The aβComAb GW-23B7 reacted strongly withintra- and extracellular material in the AD brains consistent withdystrophic neurons and processes as seen in FIG. 32A top panel. The agematched control showed positive but lighter cytoplasmic stain only inscattered neurons (FIG. 32A middle panel), and the brain from the youngcontrol with no neuropathology showed no apparent reactivity in allareas scanned (FIG. 31A bottom panel). The proved specificity of theGW-23B7 for human AD pathology was further analyzed to determine thenature of the cross-reactivity. The aβComAb GW-23B7 co-localized in thehuman AD brain not only with extracellular material of Aβ nature asdetermined by 4G8/6E10 antibodies, but interestingly with intracellularAβ in certain neurons (FIG. 32B top panel); most probably the reactivitydue to interaction with oligomeric forms of Aβ as demonstrated inimmunoblots of electron microscopy (EM) corroborated samples (FIGS. 32Cand 32D). The same AD brain showed co-localization of GW-23B7 and PHF-1in the cytoplasm of neurons (FIG. 32B middle, right panel); again thecross-reactivity was corroborated on immunoblots of EM confirmed samplesof oligomer laced human PHF (FIG. 32C, 32D). To further assesscross-reactivity with generic β-sheet in oligomeric conformers, samplesfrom a brain of an unrelated NDD Gerstmann-Straussler-Scheinker (GSS) ahuman prion disease, were included. The GW-23B7 strongly co-localizedwith the typical histopathological gliosis of prion disease where theprion particles with extremely high I3-sheet content are associated tothe GFAP in astrocytes (FIG. 32B bottom, right panel); the identity ofthe reaction to PrP^(res) was confirmed by immunoblot (FIG. 32D).

The IgM aβComAb GW-23B7 was further analyzed on histological samples ofhuman cortex and hippocampus of AD brains where most of the pathologywould be located and the primary target for potential immunotherapy. TheGW-23B7 detected, in both cortex and hippocampus, cytoplasmic changes inneurons retaining cell shape and membrane integrity through the wholespectrum to dystrophic neurons loosing membrane identity and leakingscatter punctuated material to the extracellular milieu. Processes werealso evident and potentially along axons, dendrites, budding andsynaptic zones; some of the neurons had even compromised nuclei (FIGS.33A, 33B, 33C).

The specific cross-reactivity of aβComAb GW-23B7 to potentially toxicneuropathological conformers responsible for the prion-like spread ofdisease without targeting a particular self-sequence primary structure,lead to the possibility of using GW-23B7 for immunotherapeuticalinfusion experiments in AD animal models. The mAb to be inoculatedpassed the criteria of being >99% pure IgMk pentameric (FIG. 34A),reactive with oligomeric/fibrillar forms of real human PHF inimmunoblots and detect intra and extracellular tau pathology in a humanAD brain (FIG. 34B), cross-reactive with oligomeric forms of Aβ inimmunoblots, and capable of co-localization with material related toamyloid plaques in a human AD brain (FIG. 34C).

The AD 3×Tg APP/PS1 P301L mouse model with both Aβ and tau pathology wasselected for testing. The animals were at least 16 month old to assure aflourish and fast developing Aβ and tau pathology in all animals usedfor the experiment. Two groups of ten animals each were infusedintraperitoneally (i.p.) with either aβComAb GW-23B7 in sterile salineor saline vehicle alone as per protocol in FIG. 35A. Due to the highmolecular weight of the pentameric IgM and the uncertainty of theinteraction with the blood brain barrier (BBB) two more groups of 12animals each were also infused i.p. as shown in the bottom part of theprotocol in FIG. 35A. Four animals of each group were sacrificed at 6,24 and 48 hours post-infusion respectively. Each animal was thoroughlyperfused with PBS as explained infra to devoid all brain capillaryvessels of residual immunoglobulin carrying blood. A whole hemisphere ofeach animal was homogenized and centrifuged to retain the soluble brainmaterial (FIG. 35A).

Soluble material was concentrated at least 400 times and the samplesfrom each group pooled for immunoblot analysis. FIG. 35B shows proteinreversible stain (top panel) to ascertain comparable protein load ofeach pool. The middle panel shows the anti-mouse μH chain specificreactivity before and after reduction; the intact pentameric IgM and theidentity of the reduced chain at 76 kDa molecular weight. The bottompanel shows the reactivity with anti-mouse kappa L chain antiseradetecting the intact IgMk in the same pentameric position and also thenormal polyclonal IgGk at around 150 kDa. The densitometric analysis ofeach band is shown in the graph on the right of FIG. 35B. At all timeschecked, animals infused with aβComAb GW-23B7 had a significant higherconcentration of IgM pentameric than the control group. The IgM in theGW-23B7 infused group was noticeable already at 6 hours post-infusion,peaked at 24 hours as demonstrated by both anti-μ and the anti-kappaantisera, maintaining a substantial increase after 48 hours (FIG. 35Bright graph). The control group had similar concentration of IgM at alltimes checked; and all groups infused or control had comparableconcentrations of IgG compared to the increased IgMk in the GW-23B7infused group as determined by the kappa L chains detected in bothclasses of immunoglobulins (FIG. 35B bottom panel); showing the IgMincrease in the infused animals was real and not an artifact of theprocedure. These results show IgM pentameric can go through the BBB andeventually act inside the brain.

The two groups were infused for two months as per protocol on FIG. 35A,after which all animals already 18 month old showed no difference in thelocomotor tests (FIGS. 36A-36B). The two groups were later tested at thesame time for behavioral changes determined by radial arm maze. TheGW-23B7 treated animals showed a significant cognitive rescue comparedto the control group (FIG. 35C). Immediately after, all animals weresacrificed at 19 month of age and organs and brains collected anddivided for histology and biochemistry as per standard proceduresdescribed herein.

FIG. 37A shows representative samples of hippocampus and subiculum frombrains in the GW-23B7 infused or the control groups. The strikingdifference in the mostly extracellular Aβ deposited material isreflected in the quantitation of the amyloid burden between the twogroups on FIG. 37B. On the other hand the microscopy and thequantitation analysis does not show a difference between the intensityand the number of cells with PHF-1 stain in both groups (FIG. 37A rightpanel, 37B right graph).

Conversely, the brain homogenates that would have collected the solubleor semi-soluble conformers from all parts of the brain showed adifferent panorama. The Aβ40 and Aβ42 showed a significant decline inthe GW-23B7 infused animals that can be attributed in part to asignificant reduction of recognizable aggregated Aβ (FIG. 37C three leftgraphs), a sign of toxic oligomers reduced in treated animals. Morerelevant is the specific significant drop of the toxic spreadable formof tau pTau-Thr 231 in the treated animals while the total soluble taushows only a trend to a slight decrease in the treated animals (FIG. 37Ctwo right graphs).

To assess the possible identity of the potential soluble oligomericforms of Aβ and tau which were modified by the immunotherapy, thesoluble portion of the brain homogenates was run on gels and blotted(FIG. 38A). The membrane, with representative animals from both groups,was stained with a reversible Fast Green and showed comparable loadsamong all samples and in an extended range of molecular weights (topleft panel). The same membrane was blotted with 4G8/6E10 to detectaggregated Aβ (top right panel), with PHF-1 to detect abnormal tauconformers (bottom left panel), and with the same GW-23B7 that was usedfor immunotherapeutically infuse animals (bottom right panel). Thepotential oligomeric forms to be analyzed were color coded andquantitated densitometrically after proper scan of the films. The graphresults are shown on FIG. 38B. Only one band at 110 kDa showed identityfor both Aβ and tau and was also recognized by the GW-23B7. All threeantibodies showed a significant decrease of this hetero-oligomer in thetreated animals (top row three left graphs). From 54 to 62 kDa threebands were detected by PHF-1 and showed a significant decrease in theGW-23B7 treated animals (bottom row graphs). The 54 kDa (green) decreasewas also confirmed by GW-23B7, the 57 kDa could only be measured withPHF-1 and the decrease of the 62 kDa was only significant when measureby PHF-1. A band of high molecular weight of around 190 kDa was seenonly by the GW-23B7 and significantly decreased in the treated animals.

Discussion Neurodegenerative diseases and Alzheimer's disease inparticular have been recognized and studied for more than 100 years, yetmost therapeutic approaches have yielded little or null success.Recently, immunotherapy for AD was deemed to be a potent valuableoption; however, even the most up to date trials have shown poor ordiscouraging results.

The approach for the treatment of AD described herein follows therationale that only a therapy addressing together both hallmarks of thedisease, i.e., the Aβ and tau pathologies, might have some chances ofsuccess.

The first consideration for this rationale was to have, if possible, animmune reaction to a generic trait present on both so differentpathologic conformers. It is clear now that oligomers with mobility tospread prion-like are the disease culprits more than the precipitatedamyloid Aβ in extracellular plaques and the tau-PHF inside neurons.These two extremely different neuroconformers only share in common—aswith the toxic oligomers in Parkinson and Prion diseases—the dominantβ-sheet secondary structure produced during the conversion in the brainof the self-physiological protein/peptides to pathologicoligo-conformers. An extremely difficult feat, was the development of animmunogen with no sequence similarity to any mammal protein/peptide, andpolymerization of this immunogen into extremely stable oligomers withmore than 90% β-sheet structure and no tendency to develop fibrillaryforms. This immunogen was proven to prevent pathology in three differentmouse models of AD covering the whole range of Aβ, tau and vascularamyloid pathologies. The successful immunogen was used to inoculate miceand produce hybridomas selected exclusively with at least threedifferent neuroconformers having in common only β-sheet dominantstructures in their oligomers. A few families of monoclonals thatcross-reacted with different pathological conformers were rescued andcalled anti-β-sheet secondary structure conformational monoclonalantibodies (aβComAb) to differentiate them from other anti-conformationmonoclonals raised against a specific primary and tertiary structurethat produces very limited cross-reactivity in the best of cases.

The focus of this example is aβComAb GW-23B7. This mAb has beenextensively studied and purified showing a good binding affinity tooligomeric forms of Aβ1-42 and oligomers from real PHF extracted from ahuman AD brain (FIGS. 30-33). The potential anti-β-sheetcross-reactivity was confirmed by also detecting prion oligomers and theco-localization with different pathological components in human brainsof AD and prion disease (FIGS. 32A-32D). To translate it into atherapeutic agent GW-23B7 was purified and shown to still conserve thespecific cross-reactivities (FIGS. 34A-34C).

Usually, high molecular weight bands are not detected by commercialantibodies due probably to the specific epitopes being buried in largercompact aggregates of more than one neuroconformer. On the other hand,the aβComAb GW-23B7 essentially detects the β-sheet structures inoligomers that grow by the zipper-like binding site that buries oneepitope while producing a newly-formed epitope-binding motif, ensuringthe growth of the oligomers until they get compacted, fibrillized andprecipitated as amyloid. To be able to recognize this type of oligomershas two potential advantages; the first being of diagnostic value of anactive pathology disease-spread marker, and the second, and may be laterthe most important one, the recognition of therapeutic targets that canbe used to stop and reverse a disease spread progress as it was shown inthe 3×Tg AD animal model.

Lately, there have been reports blaming high molecular weight oligomersthat are extremely toxic for rapid progression of disease and the fataldemise. The aβComAb GW-23B7 or others, alone or in combination, derivedfrom the same family of anti-secondary structure monoclonals produced asdescribed herein provide a potential solution for the treatment of themost aggressive stages of neurodegenerative diseases when spread runsout of the controls of metabolic checks and balances in the brain.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1. An antibody or binding fragment thereof comprising a heavy chainvariable region, wherein said heavy chain variable region comprises: acomplementarity-determining region 1 (H-CDR1) comprising an amino acidsequence of any one of SEQ ID NOs: 23-26, and 50, or a modified aminoacid sequence of any one of SEQ ID NOs: 23-26, and 50, said modifiedsequence containing 1, 2, or 3 amino acid residue modifications ascompared to any one of SEQ ID NOs: 23-26 and 50; acomplementarity-determining region 2 (H-CDR2) comprising an amino acidsequence of any one of SEQ ID NOs: 27-30, and 51, or a modified aminoacid sequence of any one of SEQ ID NOs: 27-30, and 51, said modifiedsequences containing 1, 2, 3, or 4 amino acid residue modifications ascompared to any one of SEQ ID NOs: 27-30, and 51; and acomplementarity-determining region 3 (H-CDR3) comprising an amino acidsequence of any one of SEQ ID NOs: 31-34, and 52, or a modified aminoacid sequence of any one of SEQ ID NO: 31-34, and 52, said modifiedsequence containing 1, 2, or 3 amino acid residue modifications ascompared to any one of SEQ ID NOs: 31-34 and
 52. 2.-4. (canceled)
 5. Theantibody or binding fragment thereof of claim 1, wherein said antibodyor binding fragment thereof is a monoclonal antibody or binding fragmentthereof.
 6. The antibody or binding fragment thereof of claim 1, whereinsaid heavy chain variable region comprises: the H-CDR1 comprising theamino acid sequence of SEQ ID NO: 23, or a modified amino acid sequencethereof, said modified amino acid sequence containing 1 or 2 amino acidresidue modifications as compared to SEQ ID NO: 23; the H-CDR2comprising the amino acid sequence of SEQ ID NO: 27, or a modified aminoacid sequence thereof, said modified amino acid sequence containing 1,2, 3, or 4 amino acid residue modifications as compared to SEQ ID NO:27; and the H-CDR3 comprising the amino acid sequence of SEQ ID NO: 31,or a modified amino acid sequence thereof, said modified amino acidsequence containing 1 or 2 amino acid modifications as compared to SEQID NO:
 31. 7. The antibody or binding fragment thereof of claim 1,wherein said heavy chain variable region comprises: the H-CDR1comprising the amino acid sequence of SEQ ID NO: 24, or a modified aminoacid sequence thereof, said modified amino acid sequence containing 1 or2 amino acid residue modifications as compared to SEQ ID NO: 24; theH-CDR2 comprising the amino acid sequence of SEQ ID NO: 28, or amodified amino acid sequence thereof, said modified amino acid sequencecontaining 1, 2, 3, or 4 amino acid residue modifications as compared toSEQ ID NO: 28; and the H-CDR3 comprising the amino acid sequence of SEQID NO: 32, or a modified amino acid sequence thereof, said modifiedamino acid sequence containing 1, 2, or 3 amino acid modifications ascompared to SEQ ID NO:
 32. 8. The antibody or binding fragment thereofof claim 1, wherein said heavy chain variable region comprises: theH-CDR1 comprising the amino acid sequence of SEQ ID NO: 25, or amodified amino acid sequence thereof, said modified amino acid sequencecontaining 1 or 2 amino acid residue modifications as compared to SEQ IDNO: 25; the H-CDR2 comprising the amino acid sequence of SEQ ID NO: 29,or a modified amino acid sequence thereof, said modified amino acidsequence containing 1, 2, 3, or 4 amino acid residue modifications ascompared to SEQ ID NO: 29; and the H-CDR3 comprising the amino acidsequence of SEQ ID NO: 33, or a modified amino acid sequence thereof,said modified amino acid sequence containing 1 or 2 amino acid residuemodifications as compared to SEQ ID NO:
 33. 9. The antibody or bindingfragment thereof of claim 1, wherein said heavy chain variable regioncomprises: the H-CDR1 comprising the amino acid sequence of SEQ ID NO:26, or a modified amino acid sequence thereof, said modified amino acidsequence containing 1 or 2 amino acid residue modifications as comparedto SEQ ID NO: 26; the H-CDR2 comprising the amino acid sequence of SEQID NO: 30, or a modified amino acid sequence thereof, said modifiedamino acid sequence containing 1, 2, 3, or 4 amino acid residuemodifications as compared to SEQ ID NO: 30; and the H-CDR3 comprisingthe amino acid sequence of SEQ ID NO: 34, or a modified amino acidsequence thereof, said modified amino acid sequence containing 1 or 2amino acid residue modifications as compared to SEQ ID NO:
 34. 10. Theantibody or binding fragment thereof of claim 1, wherein said heavychain variable region comprises: the H-CDR1 comprising the amino acidsequence of SEQ ID NO: 50, or a modified amino acid sequence thereof,said modified amino acid sequence containing 1, 2, or 3 amino acidresidue modifications as compared to SEQ ID NO: 50; the H-CDR2comprising the amino acid sequence of SEQ ID NO: 51, or a modified aminoacid sequence thereof, said modified amino acid sequence containing 1,2, 3, or 4 amino acid residue modifications as compared to SEQ ID NO:51; and the H-CDR3 comprising the amino acid sequence of SEQ ID NO: 52,or a modified amino acid sequence thereof, said modified amino acidsequence containing 1, 2, or 3 amino acid residue modifications ascompared to SEQ ID NO:
 52. 11.-18. (canceled)
 19. The antibody orbinding fragment thereof of claim 1, wherein said heavy chain variableregion further comprises human or a humanized immunoglobulin heavy chainframework regions.
 20. The antibody or binding fragment thereof of claim19, wherein said heavy chain variable region comprises an amino acidsequence that is at least 60% identical to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, and 20.21.-25. (canceled)
 26. The antibody or binding fragment thereof of claim1, wherein said antibody or binding fragment thereof is a single domainantibody.
 27. (canceled)
 28. An isolated polynucleotide encoding theantibody or binding fragment thereof of claim
 1. 29. A vector comprisingthe isolated polynucleotide of claim
 28. 30. A host cell comprising thevector of claim
 29. 31. A pharmaceutical composition comprising: theantibody or binding fragment thereof of claim 1, and a pharmaceuticalcarrier.
 32. (canceled)
 33. A method of treating a condition mediated byan amyloidogenic protein or peptide in a subject, said methodcomprising: administering to the subject the pharmaceutical compositionof claim 31, wherein said composition is administered in an amounteffective to treat the condition or one or more symptoms mediated by theamyloidogenic protein or peptide in the subject.
 34. (canceled)
 35. Themethod of claim 33, wherein the condition is selected from the groupconsisting of Alzheimer's disease (AD) and all its variations,preclinical AD, Rapid Progressive dementia, Down syndrome (DS),fronto-temporal dementia (FTD), Lewy Body Dementia (LBD), Parkinson'sdisease (PD), hereditary cerebral hemorrhage with amyloidosis (HCHWA),kuru, Creutzfeldt-Jakob disease (CJD, including familial, sporadic ornew Variant (nV) forms), chronic wasting disease (CWD) and its adaptedforms in other mammals, Gerstmann-Straussler-Scheinker disease (GSS),bovine spongiform encephalopathy (BSE) and its adapted forms in othermammals, ovine Scrapie (Sc) and its adapted forms in other mammals,Huntington's disease (HD) and all its glutamine expansion repeats, fatalfamilial insomnia, British familial dementia and all its variations,Danish familial dementia and all its variations, frontotemporal lobardegeneration associated with protein tau (FTLD-tau), frontotemporallobar degeneration associated with protein FUS (FTLD-FUS), FTD-TDP-43,Amyotrophic lateral sclerosis (ALS), FTD and ALS with all repeatexpansions due to mutations on C9orf72, Mild Cognitive Impairment (MCI),familial corneal amyloidosis, Familial corneal dystrophies, medullarythyroid carcinoma, insulinoma, type 2 diabetes, isolated atrialamyloidosis, pituitary amyloidosis, aortic amyloidosis, plasma celldisorders, familial amyloidosis, senile cardiac amyloidosis,inflammation-associated amyloidosis, familial Mediterranean fever (FMF),dialysis-associated amyloidosis, systemic amyloidosis, familial systemicamyloidosis, motor neuron disease, traumatic brain injury (TBI), adchronic traumatic encephalopathy.
 36. A method of treating a subjecthaving or at risk of having a condition mediated by a pathologicalprotein having a β-sheet secondary structure, said method comprising:administering to the subject the pharmaceutical composition of claim 31,wherein said composition is administered in an amount effective totreat, inhibit, or slow the progression of the condition or one or moresymptoms associated with the conditions mediated by the pathologicalprotein having a β-sheet secondary structure.
 37. (canceled)
 38. Amethod of diagnosing an amyloid disease in a subject, said methodcomprising: detecting, in the subject, the presence of an amyloidogenicprotein or peptide using a diagnostic reagent, wherein the diagnosticreagent comprises the antibody or binding fragment thereof of claim 1,and diagnosing the amyloid disease in the subject based on saiddetecting. 39.-40. (canceled)
 41. A method of identifying a subject'srisk for developing a condition mediated by an amyloidogenic protein orpeptide, said method comprising: detecting, in the subject, the presenceof an amyloidogenic protein or peptide using a diagnostic reagent,wherein the diagnostic reagent comprises the antibody or bindingfragment thereof of claim 1, and identifying the subject's risk ofdeveloping the condition mediated by the amyloidogenic protein orpeptide based on said detecting. 42.-43. (canceled)
 44. A diagnostic kitcomprising: the antibody or binding fragment thereof of claim 1 and adetectable label.